Pathways to Generate Hair Cells

ABSTRACT

This disclosure relates to methods and compositions for modulating (e.g., increasing) Atoh1 activity (e.g., biological activity) and/or expression (e.g., transcription and/or translation) in vivo and/or in vitro, e.g., in a biological cell and/or in a subject. The methods and compositions described herein can be used in the treatment of diseases and/or disorders that would benefit from increased Atoh1 expression in a biological cell.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.13/130,607, filed Aug. 26, 2011, which is the National Stage ofInternational Application No. PCT/US2009/065747, filed Nov. 24, 2009,and claims priority under 35 USC §119(e) to U.S. Provisional PatentApplication Ser. No. 61/117,515, filed on Nov. 24, 2008. The entirecontents of the foregoing applications are hereby incorporated byreference.

TECHNICAL FIELD

This disclosure relates to methods and compositions for modulating(e.g., increasing) Atoh1 activity (e.g., biological activity) and/orexpression (e.g., transcription and/or translation) in vivo and/or invitro, e.g., in a biological cell and/or in a subject. Morespecifically, the methods and compositions described herein can be usedin the treatment of diseases and/or disorders that would benefit fromincreased Atoh1 expression in a biological cell.

BACKGROUND

Atonal protein homologue 1 (Atoh1 or atonal) is a proneural gene thatencodes a basic helix-loop-helix (bHLH) domain-containing protein thatseems to play an important role in cell fate determination in thedevelopment of the Drosophila nervous system (Jarman et al., Cell,73:1307-1321, 1993). Atoh1 is evolutionarily conserved, with homologsidentified in Tribolium castenium (the red flour beetle), Fugu rubripes(puffer fish), chicken (Cath1), mouse (Math1), and human (Hath1)(Ben-Arie et al., Hum. Mol. Gene., 5:1207-1216, 1996). Each of thesehomologs contain a bHLH domain that is identical in length and have highsequence identity to the Atoh1 bHLH domain. For example, the Hath1 andMath1 genes are almost identical in length.

These molecules also have highly similar nucleotide sequences (86%identity) and highly similar bHLH amino acid sequences (89%). The bHLHdomain of Cath1 is 97% and 95% identical to the bHLH domain of Hath1 andMath1, respectively. The bHLH of Cath1 is 67% identical to the Atoh1bHLH domain. In contrast, the bHLH domains of other Drosophila encodedproteins share only 40-50% sequence identity.

Each of the mammalian Atoh1 homologs function as transcription factorsthat activate E box (CANNTG (SEQ ID NO:1)) dependent transcription (Arieet al., supra; Akazawa et al., J. Biol. Chem., 270:8730-8738, 1995) andfunction as critical positive regulators of cell fate determination inneural tissue and the gastrointestinal (GI) tract (Helms et al.,Development, 125:919-928, 1998; Isaka et al., Eur. J. Neurosci.,11:2582-2588, 1999; Ben-Arie et al., Development, 127:1039-1048, 2000).In addition, Atoh1 is critical for auditory hair cell development frominner ear progenitor cells, as demonstrated by the absence of auditoryhair cells in Atoh1 knockout animals (Bermingham et al., Science,284:1837-1841, 1999).

Once activated, Atoh1 transcription is self perpetuating due to thebinding of Atoh1 to the Atoh1 3′ enhancer (Helms et al., Development,127:1185-1196, 2000), and the Atoh1 promoter is switched on in Atoh1knockout mice (Bermingham et al., Science, 284:1837-1841, 1999; Tsuchiyaet al., Gastroenterology, 132:208-220, 2007). These observation indicatethat mechanisms to activate Atoh1, such as upstream regulators of Atoh1,must exist. Such upstream regulators of Atoh1 are likely to haveimportant roles in the regulation of development in the central andperipheral nervous systems and in the intestinal epithelium, all ofwhich rely on Atoh1 for differentiation.

SUMMARY

The present disclosure features methods and compositions for modulating(e.g., increasing) Atoh1 expression (e.g., transcription and/ortranslation) and/or activity (e.g., biological activity) a subjectand/or target cell.

Thus, in one aspect, the invention provides methods for treating asubject who has or is at risk of developing hearing loss or vestibulardysfunction. The methods include identifying a subject who hasexperienced, or is at risk for developing, hearing loss or vestibulardysfunction; and administering to the ear of the subject a compositioncomprising one or more compounds that increase β-catenin expression oractivity in a cell in the subject's ear; thereby treating the hearingloss or vestibular dysfunction in the subject.

In some embodiments, the subject has or is at risk for developingsensorineural hearing loss, auditory neuropathy, or both. In someembodiments, the subject has or is at risk for developing a vestibulardysfunction that results in dizziness, imbalance, or vertigo.

In some embodiments, the composition is administered systemically. Insome embodiments, the composition is administered locally to the innerear.

In some embodiments, the composition comprises a β-catenin polypeptide.In some embodiments, the composition comprises one or more Wnt/β-cateninpathway agonists. In some embodiments, the composition comprises one ormore glycogen synthase kinase 3β (GSK3β) inhibitors. In someembodiments, the composition comprises one or more casein kinase 1 (CK1)inhibitors.

In some embodiments, the methods further include administering aninhibitor of the Notch signaling pathway to the subject. In someembodiments, the inhibitor of the Notch signaling pathway is a gammasecretase inhibitor.

In some embodiments, the composition comprises a pharmaceuticallyacceptable excipient.

In another aspect, the invention provides methods for treating a subjectwho has or is at risk of developing hearing loss or vestibulardysfunction, the method comprising selecting a subject in need oftreatment, obtaining a population of cells capable of differentiatinginto hair cells, contacting the population of cells in vitro with aneffective amount of a composition comprising one or more compounds thatincrease β-catenin expression or activity for a time sufficient toinduce at least some of the cells to express one or more of p27_(kip),p75, S100A, Jagged-1, Prox1, myosin VIIa, atonal homolog 1 (Atoh1) orhomologues thereof, α9 acetylcholine receptor, espin, parvalbumin 3 andF-actin (phalloidin), optionally purifying the population of cells,e.g., to a purity of at least 50%, 60%, 70%, 80%, 90%, or more, andadministering the population of cells, or a subset thereof, to thesubjects's ear.

In some embodiments, the subject has or is at risk for developingsensorineural hearing loss, auditory neuropathy, or both.

In some embodiments, the population of cells capable of differentiatinginto auditory hair cells includes cells selected from the groupconsisting of stem cells, progenitor cells, support cells, Deiters'cells, pillar cells, inner phalangeal cells, tectal cells, Hensen'scells, and germ cells.

In some embodiments, the stem cells are adult stem cells, e.g., adultstem cells are derived from the inner ear, bone marrow, mesenchyme,skin, fat, liver, muscle, or blood, or embryonic stem cells or stemcells obtained from a placenta or umbilical cord.

In some embodiments, the progenitor cells are derived from the innerear, bone marrow, mesenchyme, skin, fat, liver, muscle, or blood.

In some embodiments, the composition comprises DNA encoding β-catenin; aβ-catenin polypeptide; one or more Wnt/β-catenin pathway agonists; oneor more glycogen synthase kinase 3β (GSK3β) inhibitors; and/or one ormore casein kinase 1 (CK1) inhibitors.

In some embodiments, administering the population of cells comprises (a)injecting the cells into the luminae of the cochlea, into the auditorynerve trunk in the internal auditory meatus, or into the scala tympanior (b) implanting the cells within a cochlea implant.

In some embodiments, the methods further include contacting the cellswith an inhibitor of the Notch signaling pathway, e.g., a gammasecretase inhibitor, e.g., one or more of an arylsulfonamide, adibenzazepine, a benzodiazepine,N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester(DAPT), L-685,458, or MK0752.

In some embodiments, the methods further include administering to theear of the subject a composition comprising one or more compoundscapable of increasing β-catenin expression or activity in a cell in thesubject's ear, e.g., DNA encoding β-catenin, a β-catenin polypeptide,one or more Wnt/β-catenin pathway agonist, one or more glycogen synthasekinase 3 (GSK3β) inhibitors, and/or one or more casein kinase 1 (CK1)inhibitors.

In some embodiments, the methods further include administering to theear of the subject a composition comprising one or more inhibitors ofthe Notch signaling pathway, e.g., a gamma secretase inhibitor.

In yet a further aspect, the invention provides methods for treating asubject who has or is at risk of developing hearing loss or vestibulardysfunction including identifying a subject who has experienced, or isat risk for developing, hearing loss or vestibular dysfunction;administering to the ear of the subject a composition comprising one ormore compounds that specifically increase β-catenin expression oractivity in a cell in the subject's ear; and administering an inhibitorof the Notch signaling pathway, e.g., a gamma secretase inhibitor, tothe subject; thereby treating the hearing loss or vestibular dysfunctionin the subject.

In some embodiments, the composition includes one or more Wnt/β-cateninpathway agonists. In some embodiments, the composition comprises one ormore glycogen synthase kinase 3β (GSK3β) inhibitors. In someembodiments, composition comprises one or more casein kinase 1 (CK1)inhibitors.

In some embodiments, the one or more CK1 inhibitors is antisense RNA orsiRNA that binds specifically to CK1 mRNA

In some embodiments, the composition comprises one or more proteasomeinhibitors.

In some aspects, the present disclosure provides methods for treating asubject or subjects that have or are at risk of developing hearing lossor vestibular dysfunction. These methods include methods for treatinghearing loss or vestibular dysfunction in the subject steps byidentifying a subject who has experienced, or is at risk for developing,hearing loss or vestibular dysfunction, and administering to the ear ofthe subject a composition comprising one or more compounds capable ofincreasing β-catenin expression or activity in a cell in the subject'sear.

In another aspect, the present disclosure provides methods of treating asubject who has or is at risk of developing hearing loss or vestibulardysfunction. These methods include selecting a subject in need oftreatment, obtaining a population of cells capable of differentiatinginto auditory hair cells, contacting the population of cells in vitrowith an effective amount of a composition comprising one or morecompounds capable of increasing β-catenin expression or activity for atime sufficient to induce at least some of the cells to express: (a) oneor more of p27_(kip), p75, S100A, Jagged-1, Prox1, myosin VIIa, atonalhomolog 1 (Atoh1) or homologues thereof, α9 acetylcholine receptor,espin, parvalbumin 3 and F-actin (phalloidin); or (b) one or more ofmyosin VIIa, atonal homolog 1 (Atoh1) or homologues thereof, andadministering the population of cells, or a subset thereof, to thesubject's ear. In some embodiments, the population of cells capable ofdifferentiating into hair cells expresses one or more of p27_(kip), p75,S100A, Jagged-1, Prox1, myosin VIIa, atonal homolog 1 (Atoh1) orhomologues thereof, α9 acetylcholine receptor, espin, parvalbumin 3 andF-actin (phalloidin).

In yet another aspect, the present disclosure provides methods ofincreasing the number of cells that express one or more of (a)p27_(kip), p75, S100A, Jagged-1, Prox1, myosin VIIa, atonal homolog 1(Atoh1) or homologues thereof, α9 acetylcholine receptor, espin,parvalbumin 3 and F-actin (phalloidin), or (b) one or more of myosinVIIa, atonal homolog 1 (Atoh1) or homologues thereof, e.g., in vitro.These methods include steps of obtaining a population of cells capableof differentiating into cells that express one or more of (a) p27_(kip),p75, S100A, Jagged-1, Prox1, myosin VIIa, atonal homolog 1 (Atoh1) orhomologues thereof, α9 acetylcholine receptor, espin, parvalbumin 3 andF-actin (phalloidin), or (b) one or more of myosin VIIa, atonal homolog1 (Atoh1) or homologues thereof, and contacting the population of cellsin vitro with an effective amount of a composition comprising one ormore compounds capable of increasing β-catenin expression or activityfor a time sufficient to increase the number of cells with thecharacteristics of auditory hair cells in the population of cells.

In a further aspect, the present disclosure provides a population ofcells in which the number of cells that express one or more of (a)p27_(kip), p75, S100A, Jagged-1, Prox1, myosin VIIa, atonal homolog 1(Atoh1) or homologues thereof, α9 acetylcholine receptor, espin,parvalbumin 3 and F-actin (phalloidin), or (b) one or more of myosinVIIa, atonal homolog 1 (Atoh1) or homologues thereof, is increased. Insome embodiments, this population of cells is obtained by obtaining apopulation of cells capable of differentiating into cells that expressone or more of (a) p27_(kip), p75, S100A, Jagged-1, Prox1, myosin VIIa,atonal homolog 1 (Atoh1) or homologues thereof, α9 acetylcholinereceptor, espin, parvalbumin 3 and F-actin (phalloidin), or (b) one ormore of myosin VIIa, atonal homolog 1 (Atoh1) or homologues thereof,contacting the population of cells in vitro with an effective amount ofa composition comprising one or more compounds capable of increasingβ-catenin expression or activity for a time sufficient to increase thenumber of cells with the characteristics of auditory hair cells in thepopulation of cells.

In some embodiments, this population of cells contacted expresses one ormore of p27kip, p75, A100AS100A, Jagged-1, Prox1, α9 acetylcholinereceptor, espin, parvalbumin 3 and F-actin (phalloidin).

In a further aspect, the present disclosure includes kits that include acomposition comprising one or more compounds capable of increasingβ-catenin expression or activity and informational material. In someembodiments, the these kits include DNA encoding β-catenin.

In an additional aspect, the present disclosure provides methods oftreating a subject who has or is at risk of developing hearing loss orvestibular dysfunction. Such methods include steps of identifying asubject who has experienced, or is at risk for developing, hearing lossor vestibular dysfunction, administering to the ear of the subject acomposition comprising one or more compounds capable of increasingβ-catenin expression or activity in a cell in the subject's ear, andadministering an inhibitor of the Notch signaling pathway to thesubject.

In some aspects, the subject selected for any of the methods disclosedherein is at risk for developing sensorineural hearing loss, auditoryneuropathy, or both. For example, the subject is at risk for developinga vestibular dysfunction that results in dizziness, imbalance, orvertigo. Alternatively or in addition, the subject can be a subject thathas been or will be treated with an orthotoxic agent

In some aspects, the methods disclosed herein effectively increases theexpression of one or more of (a) nestin, sox2, musashi, Brn3c, islet 1,Pax2, p27_(kip), p75, S100A, Jagged-1, Prox1, myosin VIIa, Atoh1 orhomologues thereof, α9 acetylcholine receptor, espin, parvalbumin 3, andF-actin (phalloidin); (b) myosin VIIa, Atoh1 in cells in the subject'sinner ear; (c) one or more of p27_(kip), p75, S100A, Jagged-1, and Prox1in cells in the subject's inner ear; (d) one or more of murine atonalgene 1 myosin VIIa, Atoh1 or homologues thereof, α9 acetylcholinereceptor, espin, parvalbumin 3, and F-actin (phalloidin) in cells in thepatient's inner ear.

In some aspects, any composition disclosed herein can be administeredsystemically, for example, using a systemic route of administration isselected from the group consisting of parenteral administration,intravenous injection, intramuscular injection, intraperitonealinjection, oral administration, lozenges, compressed tablets, pills,tablets, capsules, drops, ear drops, syrups, suspensions, emulsions,rectal administration, a rectal suppository, an enema, a vaginalsuppository, a urethral suppository, transdermal administration,inhalation, nasal sprays, and administration using a catheter or pump.

In some aspects, any composition disclosed herein can be administeredlocally to the inner ear. For example, using injection into the luminaeof the cochlea, into the auditory nerve trunk in the internal auditorymeatus, and/or into the scala tympani. Such methods can also include,for example, administered to the middle, or the inner ear, or both,e.g., using a catheter or pump.

In some aspects, any composition disclosed herein can be administered bya route of administration selected from the group consisting of anintratympanic injection, an injection into the outer, middle, or innerear, an injection through the round window of the ear, and an injectionthrough the cochlear capsule.

In some aspects, the compositions administered in the methods disclosedherein include one or more of DNA encoding β-catenin (e.g., naked DNAencoding β-catenin, plasmid expression vectors encoding β-catenin, viralexpression vectors encoding β-catenin), β-catenin polypeptides, one ormore Wnt/β-catenin pathway agonists (e.g., selected from the groupconsisting of Wnt ligands, DSH/DVL1, 2, 3, LRP6N, WNT3A, WNT5A, andWNT3A, 5A), one or more glycogen synthase kinase 3β (GSK3β) inhibitors(e.g., selected from the group consisting of lithium chloride (LiCl),Purvalanol A, olomoucine, alsterpaullone, kenpaullone,benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8),2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3 inhibitorII), 2,4-dibenzyl-5-oxothiadiazolidine-3-thione (OTDZT),(2′Z,3′E)-6-Bromoindirubin-3′-oxime (BIO), α-4-Dibromoacetophenone(i.e., Tau Protein Kinase I (TPK I) Inhibitor),2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone,N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418),indirubin-5-sulfonamide; indirubin-5-sulfonic acid(2-hydroxyethyl)-amide indirubin-3′-monoxime;5-iodo-indirubin-3′-monoxime; 5-fluoroindirubin; 5,5′-dibromoindirubin;5-nitroindirubin; 5-chloroindirubin; 5-methylindirubin,5-bromoindirubin, 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione(TDZD-8), 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3inhibitor II), 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (OTDZT),(2′Z,3′E)-6-Bromoindirubin-3′-oxime (BIO), α-4-Dibromoacetophenone(i.e., Tau Protein Kinase I (TPK I) Inhibitor),2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, (vi)N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418),H-KEAPPAPPQSpP-NH2 (L803) (SEQ ID NO: 40) and Myr-N-GKEAPPAPPQSpP-NH2(L803-mts) (SEQ ID NO: 41)), one or more anti-sense RNA or siRNA thatbind specifically to GSK3β mRNA, one or more casein kinase 1 (CK1)inhibitors (e.g., antisense RNA or siRNA that binds specifically to CK1mRNA), one or more protease inhibitors, one or more proteasomeinhibitors. The compositions and methods disclosed herein can alsofurther include the use or administration of an inhibitor of the Notchsignaling pathway (e.g., one or more of a gamma secretase inhibitor(e.g., one or more of an arylsulfonamide, a dibenzazepine, abenzodiazepine,N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester(DAPT), L-685,458, or MK0752, and an inhibitory nucleic acid includingsmall interfering RNA, an antisense oligonucleotideoligonucleotides, anda morpholino oligo oligoss). Where an inhibitor of Notch signaling isadministered, it can be administered administered systemically (e.g.,selected from the group consisting of parenteral administration,intravenous injection, intramuscular injection, intraperitonealinjection, oral administration, lozenges, compressed tablets, pills,tablets, capsules, drops, ear drops, syrups, suspensions, emulsions,rectal administration, a rectal suppository, an enema, a vaginalsuppository, a urethral suppository, transdermal administration,inhalation, nasal sprays, and administration using a catheter or pump)and or locally (e.g., locally to the ear, for example, by injection intothe luminae of the cochlea, into the auditory nerve trunk in theinternal auditory meatus, and/or into the scala tympani). In someaspects, the inhibitor of Notch signaling can be administered by a routeof administration selected from the group consisting of an intratympanicinjection, an injection into the outer, middle, or inner ear, aninjection through the round window of the ear, injection through thecochlear capsule, and/or to the middle, or the inner ear, or both usinga catheter or pump.

In some aspects, the methods disclosed herein include the use of singlecells (i.e., an isolated cell) and/or populations of cells, wherein thecell or population of cells are capable of differentiating (e.g., can,when subjected to the methods disclosed herein, differentiate into)auditory hair cells selected from the group consisting of stem cells(e.g., adult stem cells (e.g., adult stem cells obtained from the innerear, bone marrow, mesenchyme, skin, fat, liver, muscle, or blood of asubject, e.g., the subject to be treated), embryonic stem cells, or stemcells obtained from a placenta or umbilical cord), progenitor cells(e.g., progenitor cells derived from the inner ear, bone marrow,mesenchyme, skin, fat, liver, muscle, or blood), support cells, Deiters'cells, pillar cells, inner phalangeal cells, tectal cells, Hensen'scells, and germ cells.

Definitions

As used herein, “Atoh1” refers to any and all Atoh1-associated nucleicacid or protein sequences and includes any sequence that is orthologousor homologous to, or has significant sequence similarity to, an Atoh 1nucleic acid or amino acid sequence, respectively. The sequence can bepresent in any animal including mammals (e.g., humans) and insects.Examples of Atoh1 associated sequences include, but are not limited toAtoh1 (e.g., GenBank Accession Number NM_001012432.1), Hath1 (e.g.,NM_005172.1), Math1 (e.g., NM_007500.4), and Cath1 (e.g., U61149.1 andAF467292.1), as well as all other synonyms that may be used to refer tothis protein, e.g., atonal, atonal homolog 1, Ath1, and helix-loop-helixprotein Hath1. Furthermore, multiple homologous or similar sequences canexist in an animal.

As used herein, “treatment” means any manner in which one or more of thesymptoms of a disease or disorder are ameliorated or otherwisebeneficially altered. As used herein, amelioration of the symptoms of aparticular disorder refers to any lessening, whether permanent ortemporary, lasting or transient that can be attributed to or associatedwith treatment by the compositions and methods of the present invention.

The terms “effective amount” and “effective to treat,” as used herein,refer to an amount or a concentration of one or more compounds or apharmaceutical composition described herein utilized for a period oftime (including acute or chronic administration and periodic orcontinuous administration) that is effective within the context of itsadministration for causing an intended effect or physiological outcome.

Effective amounts of one or more compounds or a pharmaceuticalcomposition for use in the present invention include amounts thatpromote increased β-catenin levels (e.g., protein levels) and/oractivity (e.g., biological activity) in target cells, increasedβ-catenin levels (e.g. protein levels) and/or activity (e.g., biologicalactivity) in the nucleus of target cells, increased Atoh1 expression oractivity, and/or that promote complete or partial differentiation of oneor more cells to treat a disease that would benefit from increased Atoh1expression, e.g., prevent or delay the onset, delay the progression,ameliorate the effects of, or generally improve the prognosis of asubject diagnosed with one or more diseases that would benefit fromincreased Atoh1 expression, e.g., one or more of the diseases describedherein. For example, in the treatment of hearing impairment, a compoundwhich improves hearing to any degree or arrests any symptom of hearingimpairment would be therapeutically effective. A therapeuticallyeffective amount of a compound is not required to cure a disease butwill provide a treatment for a disease.

The term “subject” is used throughout the specification to describe ananimal, human or non-human, to whom treatment according to the methodsof the present invention is provided. Veterinary and non-veterinaryapplications are contemplated.

The term includes, but is not limited to, birds and mammals, e.g.,humans, other primates, pigs, rodents such as mice and rats, rabbits,guinea pigs, hamsters, cows, horses, cats, dogs, sheep and goats.Typical subjects include humans, farm animals, and domestic pets such ascats and dogs.

As used herein “target cell” and “target cells” refers to a cell orcells that are capable of undergoing conversion (e.g., differentiation)to or towards a cell or cells that have characteristics of auditory haircells. Target cells include, but are not limited to, e.g., stem cells(e.g., inner ear stem cells, adult stem cells, bone marrow derived stemcells, embryonic stem cells, mesenchymal stem cells, skin stem cells,and fat derived stem cells), progenitor cells (e.g., inner earprogenitor cells), support cells (e.g., Deiters' cells, pillar cells,inner phalangeal cells, tectal cells and Hensen's cells), support cellsexpressing one or more of p27_(kip), p75, S100A, Jagged-1, Prox1, and/orgerm cells. As described herein, prior to treatment with the methods,compounds, and compositions described herein, each of these target cellscan be identified using a defined set of one or more markers (e.g., cellsurface markers) that is unique to the target cell. A different set ofone or more markers (e.g., cell surface markers) can also be used toidentify target cells that have a partial or complete conversion (e.g.,partial or complete differentiation) to or towards a cell that hascharacteristics of auditory hair cells or an auditory hair cell.

Target cells can be generated from stem cells isolated from a mammal,such as a mouse or human, and the cells can be embryonic stem cells orstem cells derived from mature (e.g., adult) tissue, such as the innerear, central nervous system, blood, skin, eye or bone marrow. Unlessstated otherwise, any of the methods described below for culturing stemcells and inducing differentiation into ear cells (e.g., hair cells) canbe used.

As used herein, “β-catenin” refers to any and all β-catenin-associatednucleic acid or protein sequences and includes any sequence that isorthologous or homologous to, or has significant sequence similarity to,a β-catenin nucleic acid or amino acid sequence.

In some embodiments, β-catenin, as used herein, refers to β-catenin(e.g., mammalian β-catenin), α-catenin (e.g., mammalian α-catenin),γ-catenin (e.g., mammalian γ-catenin), δ-catenin (e.g., mammalianδ-catenin).

As used herein, “β-catenin modulating compounds” or simply “compounds”include any compound that can increase β-catenin levels (e.g., proteinlevels) and/or activity (e.g., biological activity) in target cells.Alternatively or in addition, the strategies can promote an increase inthe levels (e.g. protein levels) and/or activity (e.g., biologicalactivity) of β-catenin in the nucleus of target cells.

As used herein, the term “expression” means protein and/or nucleic acidexpression and/or protein activity.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor.

Copies of this patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIGS. 1A and 1B are images of an agarose gel showing Atoh1 and GAPDHmRNA expression in HEK and HT29 cells, respectively. “None” indicatesthat the cells were untransfected.

FIG. 1C is an image of a gel showing mRNA expression levels of Atoh1 andGAPDH in Neuro2a and neural progenitor cells transfected with Atoh1,β-catenin, or green fluorescent protein (GFP) (as shown).

FIGS. 1D and 1F are bar graphs showing relative Atoh1 expression asassessed by RT-PCR. Atoh1 levels were normalized against the S18housekeeping gene.

FIG. 1E is an image of a gel showing mRNA expression levels of Atoh1 andGAPDH in Neuro2a and neural progenitor cells transfected with siRNAtargeted against Atoh1 or β-catenin mRNA or non-targeted siRNA as acontrol.

FIG. 1G is a line graph showing luciferase reporter expression levels.\

FIG. 1H is an image of a Western blot gel showing Atoh1 and nuclearunphosphorylated β-catenin expression levels from the nuclear fraction.

FIG. 2A is a bar graph showing Atoh1 expression in HEK cells quantifiedusing real-time polymerase chain reaction (RT-PCR). Columns representthe mean of two independent experiments each performed in triplicate.Atoh1 levels are shown relative to control cells without transfectionand are normalized to S18.

FIG. 2B is a schematic representation of the Atoh1 3′ enhancer and animage of a gel showing Atoh1 bound to β-catenin, Tcf/Lef, or serum.Input (DNA without antibody precipitation) is shown as control.

FIG. 3 is an image of an immunoblot showing Atoh1 protein expression inuntransfected HEK cells and HEK cells transfected with Atoh1, β-cateninor GFP each of which was under the control of a CMV promoter.

FIGS. 4A and 4B are images of an agarose gel showing Atoh1 and GAPDHmRNA expression in Neuro2a and mouse progenitor cells derived from mouseembryonic stem (ES) cells (mES), respectively. None indicates that cellsare untransfected.

FIG. 5 is a bar graph showing Atoh1 expression in Neuro2a cellsquantified using RT-PCR. Columns represent the mean of two independentexperiments each performed in triplicate. Atoh1 levels are shownrelative to control cells without transfection and are normalized toS18.

FIG. 6 is an image of an agarose gel showing Atoh1 enhancer region fromHEK cells amplified using chromatin immunoprecipitation (ChIP).

FIGS. 7A and 7B are images of immunoblots showing β-catenin and Tcf-Lefdetection following DNA pull down. Left lanes show proteins pulled downusing probe 309 (7A) and probe 966 (7B). Center lanes show probe 309(7A) and probe 966 (7B) competition pull downs. Right lane show proteinspulled down using mutant probe 309 (7A) and mutant probe 966 (7B).

FIG. 7C is an image of gels showing Western blotting of β-catenin andTcf-Lef.

FIG. 7D is a bar graph showing the expression of Atoh1 in untransfectedNeuo2a cells and neural progenitors.

FIGS. 8A-8E are schematics showing luciferase reporter expressioncassettes encoded by the luciferase vector pGL3. (8A) control luciferasereporter expression cassette encoding a β-globin promoter (BGZA) and afirefly luciferase gene (Luc+) in the absence of a Atoh1 3′ enhancer.(8B) Wild type luciferase reporter expression cassette encoding a BGZApromoter (BGZA), Luc+, and a wild type Atoh1 3′ enhancer. (8C) Mutantluciferase reporter expression cassette encoding a BGZA promoter (BGZA),Luc+, and a Atoh1 3′ enhancer encoding a mutated first β-catenin bindingsite located at nucleotides 309-315 of AF218258. (8D) Mutant luciferasereporter expression cassette encoding a BGZA promoter (BGZA), Luc+, anda Atoh1 3′ enhancer encoding a mutated second β-catenin binding sitelocated at nucleotides 966-972 of AF218258. (8E) Mutant luciferasereporter expression cassette encoding a BGZA promoter (BGZA), Luc+, anda Atoh1 3′ enhancer encoding mutated first and second β-catenin bindingsites at nucleotides 309-315 and 966-972 of AF218258. Nucleotidesencoded by the first and second β-catenin binding sites at nucleotides309-315 and 966-972 of AF218258 are shown in upper case font. *indicates a mutated nucleotide. Nucleotides shown with * are mutantnucleotides.

FIG. 9 is a bar graph showing relative luciferase expression in murineNeuro2a cells alone (open bars) or in the presence of β-catenin (solidbars). Cells were transfected with luciferase constructs (A)-(E)depicted in FIGS. 8A-8E.

FIGS. 10A, C, and E are images of gels showing the expression levels ofβ-catenin, Atoh1, and β-actin following treatment of cells with theγ-secretase inhibitor DAPT (used at 10 μM and 50 μM), GSK3β inhibitor,and/or siRNA targeted against β-catenin.

FIG. 10B is a bar graph showing the effect of two siRNAs directedagainst β-catenin as evaluated by RT-PCR.

FIG. 10D is a bar graph showing data collected using Pofut1−/− cells inwhich Notch signaling is inhibited.

FIG. 10F is an image of a gel showing β-catenin expression in cellsfollowing treatment with β-catenin agonists and Notch signalinginhibitors.

FIGS. 11A-11C are images of inner ear stem cells expressing fluorescentmarkers. (11A) Cells infected with adenoviruses encoding GFP. Left panelshows inner ear stem cells expressing green fluorescent protein (GFP);center panel shows cells stained with the nuclear stain4′-6-Diamidino-2-phenylindole (DAPI-blue); right panel shows a merge ofthe left and center panels. (11B) and (11C) left panels show cellsstained for myosin VIIa (red); second panels show Atoh1-nGFP positivecells (green); third panels show cells stained with DAPI; right panelsshow merged cells (red, green and blue). Triple stained cells are shownwith arrows. (11B) cells infected with empty adenovirus vector. (11C)cells infected with adenoviruses encoding human β-catenin.

FIG. 11D is a bar graph showing quantification of Atoh1 and myosin VIIadouble stained cells. Data represents three independent experiments inwhich 5000 cells were counted.

FIGS. 12A and 12B are images of inner ear stem cells expressingβ-catenin-IRES-DsRed (12A) and IRES-DsRed in the absence of β-catenin(12B). (i) shows cells expressing β-catenin-IRES-DsRed or IRES-DsRed(red). One cell is shown for both (12A) and (12B). (ii) shows Atoh1expression (green) in the same field of view as (i). (iii) shows a phasecontrast image of the same field of view as (i) and (ii). (iv) shows amerged image of (i), (ii), and (iii). Co-stained cells are shown witharrows.

FIGS. 13A-13D are images of hair cells in the organ of corti dissectedat E16 in Atoh1-nGFP mice. (13A) untreated control hair cells; (13B)hair cells infected with empty adenoviral vector for 5 days; (13C and D)hair cells infected with adenovirus encoding β-catenin for 5 days. Greencells are Atoh-1 positive hair cells.

FIGS. 14A and 14B are images of different organs of corti dissected fromAtoh1-nGFP mice. (14A) shows a dissected organ of corti 2 days postinfected with β-catenin. (14B) shows an uninfected organ of corti.

FIGS. 15A and 15B are images showing putative WNT/β-catenin signalingpathways. 15B illustrates regulation of Atoh1 by β-catenin according tothe data presented herein.

DETAILED DESCRIPTION

The present disclosure provides, inter alia, methods and pharmaceuticalcompositions for treating subjects for the conditions noted below.Accordingly, the present disclosure is based, at least in part, on thediscovery that differentiation of a cell to or towards a mature cell ofthe inner ear, e.g., an auditory hair cell can be promoted throughβ-catenin-dependent WNT signaling. In other words, the presentdisclosure provides methods and compositions relating to theWNT/β-catenin signaling pathway for generating cells that havecharacteristics of auditory hair cells.

While the treatment methods are not limited to those in which particularunderlying cellular events occur, the present compounds and compositionsmay increase the expression of an Atoh1 gene in a subject and/or targetcell.

As shown herein, β-catenin, the intracellular mediator of the canonicalWnt signaling pathway, is capable of increasing Atoh1 expression in abiological cell. Characterization of this effect revealed that β-cateninincreases Atoh1 expression through a direct interaction with twodistinct β-catenin binding domains encoded in the Atoh1 3′ enhancerregion (e.g., at nucleotides 309-315 and nucleotides 966-972 of GenBankAccession No. AF218258 (e.g., AF218258.1; G17677269)). These twoβ-catenin binding domains also interact with T-cell factor (TCF) andlymphoid enhancer-binding protein (LEF), which are transcription factorsthat normally maintain target genes of the WNT signaling pathway in arepressed state by interacting, in combination with other co-repressors,with the promoter or enhancer regions of Wnt target genes. Thus, thedata presented herein demonstrates that β-catenin serves as an upstreamregulator of Atoh1. Additionally, the data presented herein demonstratesthat β-catenin dependent Atoh1 expression promotes the differentiationof inner ear progenitor cells to or towards cells that havecharacteristics of auditory hair cells.

Catenins

Catenins are a group of proteins that are commonly found in complex withcadherin cell adhesion molecules, e.g., in animal cells. Four cateninshave been identified to date, namely: α-catenin, β-catenin, δ-catenin,and γ-catenin.

α-catenin is an actin-binding protein at the adherens junction, that hasoverall similarity to vinculin, another actin-binding protein present atadhesional complexes. α-catenin is about 100 kDa (e.g., 102 kDa) asdetected by Western Blotting (see, e.g., Nagafuchi et al., Cell,65:849-857, 1991). α-catenin is detectable by Western blotting using,e.g., anti-alpha catenin monoclonal antibody available from GenWay(e.g., catalogue number 20-272-191447).

β-catenin is capable of binding to the subdomain of some cadherins andis implicated in the WNT signaling pathway. The ability of β-catenin tobind to other proteins is regulated by tyrosine kinases and serinekinases such as GSK-3 (see, e.g., Lilien et al., Current Opinion in CellBiology, 17:459-465, 2005). β-catenin is about a 80-100 kDa (e.g., 88kDa-92 kDa, e.g., 92 kDa) as detected by Western Blotting. β-catenin isdetectable by Western blotting using, e.g., anti-beta catenin monoclonalantibody available from Abcam (e.g., catalogue number Ab2982).

δ-catenin (e.g., δ1-catenin and δ2-catenin) is a member of a family ofproteins with ten armadillo repeats (the p120 catenin subfamily ofcatenins). δ-catenin is expressed predominantly in neural tissue whereit interacts with presenilins (see, e.g., Israely et al., CurrentBiology, 14:1657-1663, 2004 and Rubio et al., Mol. And Cell. Neurosci.,4:611-623, 2005). δ-catenin is about a 100-150 kDa (e.g., about 125 kDa)as detected by Western Blotting. δ1-catenin is detectable by Westernblotting using, e.g., anti-delta catenin antibody available from SigmaAldrich (e.g., catalogue number C4989). δ2-catenin is detectable byWestern blotting using, e.g., anti-delta catenin antibody available fromAbcam (e.g., catalogue number ab54578).

γ-catenin is commonly found as a component of desmosomes and can bind todesmoglein I (see e.g., Franke et al., Proc. Natl. Acad. Sci. USA.,86:4027-31, 1989). γ-catenin is about a 80-100 kDa (e.g., about 80 kDa)as detected by Western Blotting. γ-catenin is detectable by Westernblotting using, e.g., anti-gamma catenin monoclonal antibody availablefrom Abcam (e.g., catalogue number Ab11799).

WNT/β-Catenin Signaling

The expression of bHLH transcription factors, such as Atoh1, is partlyregulated by various components of the Notch pathway. However, Notch maybe only a part of the complex regulatory circuits governing the timingand amount of bHLH transcription factor expression as well as the tissuespecificity of expression.

WNT signaling pathways (see, e.g., FIG. 14) play a key role in earlydevelopment of several tissues, including but not limited to, forexample, the intestinal epithelium and the inner ear (Clevers, Cell,127:469-480, 2006; Ohyama et al., Development, 133:865-875, 2006; Pintoet al., Exp. Cell. Res., 306:357-363, 2005; Stevens et al., Dev. Biol.,261:149-164, 2003; van ES et al., Nat. Cell. Biol., 7:381-386, 2005; vanES et al., Nature, 435:959-963, 2005). Furthermore, disruption of Wntsignaling prevents intestinal epithelial differentiation to mature celltypes accompanied by decreased Atoh1 expression (Pinto et al., supra).

WNTs are secreted cysteine-rich glycoproteins that act as short-rangeligands to locally activate receptor-mediated signaling pathways. Inmammals, 19 members of the WNT protein family have been identified. WNTsactivate more than one signaling pathway (Veerman et al., Dev. Cell.,5:367-377, 2003) including both β-catenin-dependent andβ-catenin-independent pathways. The best understood of the WNT-activatedpathways, however, is the WNT/β-catenin pathway, and the list ofproteins identified as being involved in the WNT/β-catenin pathway isextensive and expanding.

Wnt signaling is transduced intracellularly by the frizzled (Fzd) familyof receptors (Hendrickx and Leyns, Dev. Growth Differ., 50:229-243,2008). Activation of the WNT/β-catenin pathway leads to an increase inthe post-translational stability of β-catenin. As β-catenin levels rise,it accumulates in the nucleus, where it interacts and forms a complexwith DNA-bound TCF and LEF family members to activate the transcriptionof target genes. Conversely, in the absence of WNT signaling, β-cateninis recruited to a destruction complex containing adenomatous polyposiscoli (APC) and AXIN, which together serve to facilitate thephosphorylation of β-catenin by casein kinase 1 (CK1) and then glycogensynthase kinase 3 (GSK3). This process leads to the ubiquitination andproteosomal degradation of β-catenin. As a result, in the absence of WNTsignaling, cells maintain low cytoplasmic and nuclear β-catenin levels.Some β-catenin is spared from proteosomal degradation through anassociation with cadherins at the plasma membrane (Nelson et al.,Science, 303, 1483-1487, 2004).

β-catenin expression is involved in maintaining the balance between stemcell proliferation and stem cell differentiation (Chenn and Walsh,Science, 297:365-369, 2002). A role for β-catenin in the development ofmouse auditory epithelia has also been described and it has been shownthat β-catenin expression was linked with auditory epithelia developmentin mouse models (Takebayashi et al., Acta. Otolaryngol Suppl.,551:18-21, 2004). Other studies also support a role for β-catenin inpromoting cell proliferation in the developing auditory epithelia ofmice (Takebyashi et al., Neuroreport, 16:431-434, 2005; Warchol, J.Neurosci., 22:2607-2616, 2002) and rat utricles (Kim et al., Acta.Otolaryngol Suppl., 551:22-25, 2004). A further study performed in ratembryos also reports that suppression of β-catenin using antisensetechnology reduced the number of cells in the otic cup, which theauthors concluded demonstrated that β-catenin plays a role in cellproliferation in the otic placodes and in differentiation in acousticneurons within the acoustic neural crest complex (Matsuda and Keino,Anat. Embryol. (Berl)., 202:39-48, 2000). In addition, it is reportedthat the Wnt/β-catenin pathway is involved in defining and maintainingthe sensory/neurosensory boundaries in the cochlea duct (Stevens et al.,Dev. Biol., 261:149-164, 2003). Together, previously published dataindicated that β-catenin is involved in promoting stem cellproliferation, not differentiation.

Methods of Treatment

In some embodiments, the present disclosure provides novel therapeuticstrategies for treating diseases that would benefit from an increase inAtoh1 expression and/or activity. In some embodiments, such strategiescan promote an increase in the levels (e.g., protein levels) and/oractivity (e.g., biological activity) of β-catenin in target cells,thereby promoting differentiation of a target cell to or towards amature cell of the inner ear, e.g., an auditory hair cell. Alternativelyor in addition, the strategies can promote an increase in the levels(e.g. protein levels) and/or activity (e.g., biological activity) ofβ-catenin in the nucleus of target cells, thereby promotingdifferentiation of a target cell to or towards a mature cell of theinner ear, e.g., an auditory hair cell.

In some embodiments, the methods and compositions described hereinpromote differentiation of target cells to or towards mature cells ofthe inner ear, e.g., auditory hair cells without promoting substantialcellular proliferation. In some embodiments, 0, 0.5, 1, 3, 5, 10, 15,20, 25, 30, 40, or 50% of the target cells undergo proliferation upontreatment with the methods and compositions described herein.

Compositions and Methods for Modulating β-Catenin Expression

In some embodiments, the present disclosure includes the use ofcompounds, compositions (referred to collectively herein as β-cateninmodulating compounds) and methods that increase the levels (e.g.,protein levels) and/or activity (e.g., biological activity) of β-cateninin target cells. Exemplary β-catenin modulating compounds and methodsinclude, but are not limited to compositions and methods for increasingβ-catenin expression (e.g., transcription and/or translation) or levels(e.g., concentration) in target cells include the use of:

(i) DNA encoding β-catenin. β-catenin can be expressed using one or moreexpression constructs. Such expression constructs include, but are notlimited to, naked DNA, viral, and non-viral expression vectors).Exemplary β-catenin nucleic acid sequences that may be usefullyexpressed include, but are not limited to, for example, NM_001098209(e.g., NM_001098209.1), GI:148233337, NM_001904 (e.g., NM_001904.3),GI:148228165, NM_001098210 (e.g., NM_001098210.1), GI:148227671,NM_007614 (e.g., NM_007614.2), GI:31560726, NM_007614 (e.g.,NM_007614.2), and GI:31560726.

In some embodiments, β-catenin nucleic acid can include nucleic acidencoding α-catenin (e.g., NM_001903.2), δ-catenin (e.g., NM_001085467.1(δ1) and NM_01332.2 (S2)), and γ-catenin (e.g., AY243535.1 andGI:29650758)

In some embodiments, DNA encoding β-catenin can be an unmodified wildtype sequence. Alternatively, DNA encoding β-catenin can be modifiedusing standard molecular biological techniques. For example, DNAencoding β-catenin can be altered or mutated, e.g., to increase thestability of the DNA or resulting polypeptide. Polypeptides resultingfrom such altered DNAs will retain the biological activity of wild typeβ-catenin. In some embodiments, DNA encoding β-catenin can be altered toincrease nuclear translocation of the resulting polypeptide. In someembodiments, DNA encoding β-catenin can be modified using standardmolecular biological techniques to include an additional DNA sequencethat can encode one or more of, e.g., detectable polypeptides, signalpeptides, and protease cleavage sites.

(ii) β-catenin encoding polypeptides. Exemplary useful β-cateninpolypeptides include, but are not limited to, for example, NP_001091679(e.g., NP_001091679.1), GI:148233338, NP_001895 (e.g., NP_001895.1),GI:4503131, NP_001091680 (e.g., NP_001091680.1), GI:148227672, NP_031640(e.g., NP_031640.1), and GI:6671684. Such β-catenin encodingpolypeptides can be used in combination with compositions to enhanceuptake of the polypeptides into biological cells. In some embodiments,β-catenin encoding polypeptides can be mutated to include amino acidsequences that enhance uptake of the polypeptides into a biologicalcell. In some embodiments, β-catenin encoding polypeptides can bealtered or mutated to increase the stability and/or activity of thepolypeptide (e.g., β-catenin point mutants. In some embodiments,β-catenin encoding polypeptides can be altered to increase nucleartranslocation of the polypeptide. In some embodiments, alteredpolypeptides will retain the biological activity of wild type β-catenin.

In some embodiments, useful β-catenin nucleic acid sequences andβ-catenin encoding polypeptides include modified β-catenin nucleic acidsequences and β-catenin encoding polypeptides. Such modified β-cateninnucleic acid sequences and β-catenin encoding polypeptides can benucleic acids and/or polypeptide having sequences that are substantiallyidentical to the nucleic acid or amino acid sequences of NM_001098209(e.g., NM_001098209.1), GI:148233337, NM_001904 (e.g., NM_001904.3),GI:148228165, NM_001098210 (e.g., NM_001098210.1), GI:148227671,NM_007614 (e.g., NM_007614.2), GI:31560726, NM_007614 (e.g.,NM_007614.2), GI:31560726, NP_001091679 (e.g., NP_001091679.1),GI:148233338, NP_001895 (e.g., NP_001895.1), GI:4503131, NP_001091680(e.g., NP_001091680.1), GI:148227672, NP_031640 (e.g., NP_031640.1), andGI:6671684. In some embodiments, useful β-catenin nucleic acid sequencescan be 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% homologousto NM_001098209 (e.g., NM_001098209.1), GI:148233337, NM_001904 (e.g.,NM_001904.3), GI:148228165, NM_001098210 (e.g., NM_001098210.1),GI:148227671, NM_007614 (e.g., NM_007614.2), GI:31560726, NM_007614(e.g., NM_007614.2), and GI:31560726. In some embodiments, usefulβ-catenin encoding polypeptides sequences can be 50%, 60%, 70%, 80%,85%, 90%, 95%, 98%, 99%, or 100% homologous to NP_001091679 (e.g.,NP_001091679.1), GI:148233338, NP_001895 (e.g., NP_001895.1),GI:4503131, NP_001091680 (e.g., NP_001091680.1), GI:148227672, NP_031640(e.g., NP_031640.1), and GI:6671684. In some embodiments, moleculesencoded by useful modified β-catenin nucleic acid sequences andβ-catenin encoding polypeptide sequences will possess at least a portionof the activity (e.g., biological activity) of the molecules encoded bythe corresponding, e.g., unmodified β-catenin nucleic acid sequences andβ-catenin encoding polypeptide sequences. For example, molecules encodedby modified β-catenin nucleic acid sequences and β-catenin encodingpolypeptides can retain 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, or100% of the activity (e.g., biological activity) of the moleculesencoded by the corresponding, e.g., unmodified β-catenin nucleic acidsequences and β-catenin encoding polypeptide sequences. The methodsrequired to assess the activity of β-catenin or a β-catenin-likemolecule are described herein.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, or 100% of the length of thereference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thedetermination of percent identity between two amino acid sequences isaccomplished using the BLAST 2.0 program. Sequence comparison isperformed using an ungapped alignment and using the default parameters(Blossom 62 matrix, gap existence cost of 11, per residue gapped cost of1, and a lambda ratio of 0.85). The mathematical algorithm used in BLASTprograms is described in Altschul et al. (Nucleic Acids Res.25:3389-3402, 1997). Useful β-catenin encoding polypeptide sequences orpolypeptide fragments can have up to about 20 (e.g., up to about 10, 5,or 3) amino acid deletions, additions, or substitutions, such asconservative substitutions, to be useful for the compositions andmethods described herein. Conservative amino acid substitutions areknown in the art.

(iii) Wnt/β-catenin pathway agonists. In some embodiments, β-cateninlevels (e.g., protein levels) and/or activity (e.g., biologicalactivity) can be modulated (e.g., increased) using compounds orcompositions that target one or more components of the WNT/β-cateninpathway. For example, suitable compounds or compositions can target two,three, four, five or more components of the WNT/β-catenin pathway. Insome embodiments, components with opposing effects on β-catenin levels(e.g., protein levels) and/or activity (e.g., biological activity) canbe targeted. For example, a first component that increases β-cateninlevels (e.g., protein levels) and/or activity (e.g., biologicalactivity) can be targeted in combination with a second target thatinhibits β-catenin levels (e.g., protein levels) and/or activity (e.g.,biological activity). In this example, the first target would beactivated and the second target would be inhibited.

Exemplary useful β-catenin pathway agonists increase β-cateninexpression (e.g., transcription and/or translation), levels (e.g.,concentration), or activity by acting on one or more components of theWnt/β-catenin signaling pathway. For example, suitable Wnt/β-cateninpathway agonists can act indirectly (e.g., on upstream modulators orinhibitors ofβ-catenin or on components of cellular transcriptionmachinery), by increasing the stability of β-catenin (e.g., bydecreasing the degradation of β-catenin, such as through the inhibitionof casein kinase 1 (CK1) and glycogen synthase kinase 3β (GSK3β)),and/or by promoting the release of sequestered endogenous intracellularβ-catenin. Exemplary Wnt/β-catenin pathway agonists include, but are notlimited to, e.g., Wnt ligands, DSH/DVL1, 2, 3, LRP6AN, WNT3A, WNTSA, andWNT3A, 5A. Additional Wnt/β-catenin pathway activators and inhibitorsare reviewed in the art (Moon et al., Nature Reviews Genetics,5:689-699, 2004). In some embodiments, suitable Wnt/β-catenin pathwayagonists can include antibodies and antigen binding fragments thereof,and peptides that bind specifically to frizzled (Fzd) family ofreceptors.

(iv) Kinase inhibitors, e.g., casein kinase 1 (CK1) and glycogensynthase kinase 3β (GSK3β) inhibitors. In some embodiments, usefulkinase inhibitors can increase β-catenin levels by reducing thedegradation of β-catenin. In some embodiments, exemplary useful kinaseinhibitors, e.g., GSK3β inhibitors include, but are not limited to,lithium chloride (LiCl), Purvalanol A, olomoucine, alsterpaullone,kenpaullone, benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8),2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3 inhibitorII), 2,4-dibenzyl-5-oxothiadiazolidine-3-thione (OTDZT),(2′Z,3′E)-6-Bromoindirubin-3′-oxime (BIO), α-4-Dibromoacetophenone(i.e., Tau Protein Kinase I (TPK I) Inhibitor),2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone,N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418), andindirubins (e.g., indirubin-5-sulfonamide; indirubin-5-sulfonic acid(2-hydroxyethyl)-amide indirubin-3′-monoxime;5-iodo-indirubin-3′-monoxime; 5-fluoroindirubin; 5, 5′-dibromoindirubin;5-nitroindirubin; 5-chloroindirubin; 5-methylindirubin,5-bromoindirubin), 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione(TDZD-8), 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3inhibitor II), 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (OTDZT),(2′Z,3′E)-6-Bromoindirubin-3′-oxime (BIO), α-4-Dibromoacetophenone(i.e., Tau Protein Kinase I (TPK I) Inhibitor),2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, (vi)N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418), andH-KEAPPAPPQSpP-NH2 (L803) (SEQ ID NO: 40) or its cell-permeablederivative Myr-N-GKEAPPAPPQSpP-NH2 (L803-mts) (SEQ ID NO: 41). OtherGSK3β inhibitors are disclosed in U.S. Pat. Nos. 6,417,185; 6,489,344;6,608,063 and Published U.S. Applications Nos. 690497, filed Oct. 20,2003; 468605, filed Aug. 19, 2003; 646625, filed Aug. 21, 2003; 360535,filed Feb. 6, 2003; 447031, filed May 28, 2003; and 309535 filed Dec. 3,2002. In some embodiments, suitable kinase inhibitors can include RNAiand siRNA designed to decrease GSK3β and/or CK1 protein levels. In someembodiments, useful kinase inhibitors include FGF pathway inhibitors. Insome embodiments, FGF pathway inhibitors include, for example, SU5402.

(v) Protease inhibitors and Proteasome inhibitors. In some embodiments,useful protease inhibitors can increase β-catenin levels by reducing thedegradation of β-catenin. Suitable protease inhibitors are known in theart (see e.g., Shargel et al., Comprehensive Pharmacy Review, FifthEdition, published by Lippincott Williams, and Wilkins, at, e.g., pages373 and 872-874). In some embodiments, useful protease inhibitors caninclude, for example, natural protease inhibitors, synthetic proteaseinhibitors, antiretroviral protease inhibitors, and protease inhibitorcocktails.

In some embodiments, useful protease inhibitors can include inhibitorsof the proteasome or proteasome inhibitors. Suitable proteasomeinhibitors include, but are not limited to, for example, Velcade® (e.g.,bortezomib, Millenium Pharmaceuticals), MG132 (Calbiochem), lactacystin(Calbiochem), and proteasome inhibitor (PSI). In some embodiments,useful protease inhibitors can include inhibitors of the ubiquitinpathway.

(vi) Any combination of (i)-(v).

(vii) Any combination of (i)-(v) in combination with an inhibitor of theNotch signaling pathway, e.g., a gamma-secretase inhibitor or inhibitorynucleic acid. Exemplary gamma secretase inhibitors include, but are notlimited to, e.g., arylsulfonamides, dibenzazepines, benzodiazepines,N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester(DAPT), L-685,458, or MK0752. Other exemplary Notch pathway inhibitorsand methods for identifying inhibitors of the Notch signaling pathwayare disclosed in, e.g., PCT/US2007/084654, U.S. P.G. Pub. No.2005/0287127, and U.S. application Ser. No. 61/027,032.

In some embodiments, the present disclosure provides methods whereby:

(a) one or more β-catenin modulating compounds are administered to asubject, e.g., to the ear of a subject (direct therapy);

(b) one or more target cells are contacted, e.g., in vitro, with one ormore β-catenin modulating compounds to promote complete or partialconversion (e.g., differentiation) of those cells to or toward a maturecell type, e.g., a hair cell.

(c) one or more target cells that have been treated according to method(b) (e.g., one or more cells resulting from method (b)) is administeredto a subject, e.g., to the ear of a subject (cell therapy); and

(d) methods whereby one or more target cells that have been treatedaccording to method (b) (e.g., one or more cells resulting from method(b)) are administered to a subject in combination with one or moreβ-catenin modulating compounds administered to a subject, e.g., to theear of a subject (combination therapy).

Subject Selection

It is widely accepted that although cells capable of generating haircells are present in the inner ear, natural hair cell regeneration inthe inner ear is low (Li et al., Trends Mol. Med., 10, 309-315 (2004);Li et al., Nat. Med., 9, 1293-1299 (2003); Rask-Andersen et al., Hear.Res., 203, 180-191 (2005)). As a result, lost or damaged hair cells maynot be adequately replaced by natural physiological processes (e.g.,cell differentiation), and a loss of hair cells occurs. In manyindividuals, such hair cell loss can result in, e.g., sensorineuralhearing loss, hearing impairment, and imbalance disorders. Therapeuticstrategies that increase the number of hair cells in the inner ear willbenefit a subject with hair cell loss, e.g., with one or more of theseconditions.

The importance of Atoh1 in hair cell genesis is well documented. Forexample, Atoh1 is required for hair cell development and thedifferentiation of inner ear progenitor cells to inner ear support cellsand/or hair cells (Bermingham et al., Science, 284:1837-1841, 1999). Inaddition, adenovirus mediated Math1 overexpression in the endolymph ofthe mature guinea pig results in the differentiation of non-sensorycells in the mature cochlea into immature hair cells (Kawamoto et al.,The Journal of Neuroscience, 23:4395-4400, 2003;). The implications ofthese studies are twofold. First, they demonstrate that non-sensorycells of the mature cochlea retain the ability to differentiate intosensory cells, e.g., hair cells. Second, they demonstrate that Math1overexpression is necessary and sufficient to direct hair celldifferentiation from non-sensory cells. A later study furthered thesefindings by demonstrating that adenovirus mediated Atoh1 overexpressioninduces hair cell regeneration and substantially improves hearingthresholds in an experimentally deafened animal model (Izumikawa et al.,Nat. Med., 11:271-276, 2005).

In some embodiments, the methods, compounds, and compositions describedherein can be used for treating subjects who have, or who are at riskfor developing, an auditory disorder resulting from a loss of auditoryhair cells, e.g., sensorineural hair cell loss.

Subjects with sensorineural hair cell loss experience the degenerationof cochlea hair cells, which frequently results in the loss of spiralganglion neurons in regions of hair cell loss. Such subjects may alsoexperience loss of supporting cells in the organ of Corti, anddegeneration of the limbus, spiral ligament, and stria vascularis in thetemporal bone material.

In some embodiments, the present invention can be used to treat haircell loss and any disorder that arises as a consequence of cell loss inthe ear, such as hearing impairments, deafness, and vestibulardisorders, for example, by promoting differentiation (e.g., complete orpartial differentiation) of one or more cells into one or more cellscapable of functioning as sensory cells of the ear, e.g., hair cells.

In some embodiments, the methods include steps of selecting a subject atrisk of hair cell loss and/or a subject with hair cell loss.Alternatively or in addition, the methods include steps of selecting asubject at risk of sensorineural hearing loss and/or a subject withsensorineural hearing loss. Any subject experiencing or at risk fordeveloping hearing loss is a candidate for the treatment methodsdescribed herein. A human subject having or at risk for developing ahearing loss can hear less well than the average human being, or lesswell than a human before experiencing the hearing loss. For example,hearing can be diminished by at least 5, 10, 30, 50% or more.

In some embodiments, the subject can have sensorineural hearing loss,which results from damage or malfunction of the sensory part (thecochlea) or the neural part (the auditory nerve) of the ear, orconductive hearing loss, which is caused by blockage or damage in theouter and/or middle ear. Alternatively or in addition, the subject canhave mixed hearing loss caused by a problem in both the conductivepathway (in the outer or middle ear) and in the nerve pathway (the innerear). An example of a mixed hearing loss is a conductive loss due to amiddle-ear infection combined with a sensorineural loss due to damageassociated with aging.

In some embodiments, the subject can be deaf or have a hearing loss forany reason, or as a result of any type of event. For example, a subjectcan be deaf because of a genetic or congenital defect; for example, ahuman subject can have been deaf since birth, or can be deaf orhard-of-hearing as a result of a gradual loss of hearing due to agenetic or congenital defect. In another example, a human subject can bedeaf or hard-of-hearing as a result of a traumatic event, such as aphysical trauma to a structure of the ear, or a sudden loud noise, or aprolonged exposure to loud noises. For example, prolonged exposures toconcert venues, airport runways, and construction areas can cause innerear damage and subsequent hearing loss.

In some embodiments, a subject can experience chemical-inducedototoxicity, wherein ototoxins include therapeutic drugs includingantineoplastic agents, salicylates, quinines, and aminoglycosideantibiotics, contaminants in foods or medicinals, and environmental orindustrial pollutants.

In some embodiments, a subject can have a hearing disorder that resultsfrom aging. Alternatively or in addition, the subject can have tinnitus(characterized by ringing in the ears).

In some embodiments, a subject suitable for the treatment using themethods and β-catenin modulating compounds featured in this disclosurecan include a subject having a vestibular dysfunction, includingbilateral and unilateral vestibular dysfunction. Vestibular dysfunctionis an inner ear dysfunction characterized by symptoms that includedizziness, imbalance, vertigo, nausea, and fuzzy vision and may beaccompanied by hearing problems, fatigue and changes in cognitivefunctioning. Vestibular dysfunction can be the result of a genetic orcongenital defect; an infection, such as a viral or bacterial infection;or an injury, such as a traumatic or nontraumatic injury. Vestibulardysfunction is most commonly tested by measuring individual symptoms ofthe disorder (e.g., vertigo, nausea, and fuzzy vision).

In some embodiments, the methods and β-catenin modulating compoundsprovided herein can be used prophylactically, such as to prevent hearingloss, deafness, or other auditory disorders associated with loss ofinner ear function. For example, a composition containing one or morecompounds can be administered with a second therapeutic, such as atherapeutic that may affect a hearing disorder. Such ototoxic drugsinclude the antibiotics neomycin, kanamycin, amikacin, viomycin,gentamycin, tobramycin, erythromycin, vancomycin, and streptomycin;chemotherapeutics such as cisplatin; nonsteroidal anti-inflammatorydrugs (NSAIDs) such as choline magnesium trisalicylate, diclofenac,diflunisal, fenoprofen, flurbiprofen, ibuprofen, indomethacin,ketoprofen, meclofenamate, nabumetone, naproxen, oxaprozin,phenylbutazone, piroxicam, salsalate, sulindac, and tolmetin; diuretics;salicylates such as aspirin; and certain malaria treatments such asquinine and chloroquine. For example, a human undergoing chemotherapycan be treated using compounds and methods described herein. Thechemotherapeutic agent cisplatin, for example, is known to cause hearingloss. Therefore, a composition containing one or more compounds can beadministered with cisplatin therapy to prevent or lessen the severity ofthe cisplatin side effect. Such a composition can be administeredbefore, after and/or simultaneously with the second therapeutic agent.The two agents can be administered by different routes ofadministration.

In some embodiments, the treatment of auditory hair cell loss includessteps whereby one or more β-catenin modulating compounds areadministered to a subject to promote the formation of auditory haircells (e.g., an inner ear and/or outer ear hair cells) and/or increasethe number of hair cells (e.g., an inner ear and/or outer ear haircells) in the ear of a subject by promoting complete or partial haircell differentiation from non-hair cell types naturally present in theinner ear of a subject. This method of treatment is referred to asdirect therapy.

In some embodiments, the treatment of auditory hair cell loss includessteps whereby one or more target cells are contacted, e.g., in vitro,with one or more β-catenin modulating compounds to promote complete orpartial differentiation of those cells to or toward a mature cell typeof the inner ear, e.g., a hair cell (e.g., an inner ear and/or outer earhair cell).

Alternatively or in addition, the methods include steps whereby one ormore target cells that have been contacted with one or more β-cateninmodulating compounds, e.g., in vitro, are administered to the ear (e.g.,the inner ear) of the subject. This method of therapy is referred to ascell therapy.

In some embodiments, the methods include steps whereby one or moretarget cells that have been contacted with one or more β-cateninmodulating compounds, e.g., in vitro are administered to the ear (e.g.,inner ear) of a subject in combination with one or more β-cateninmodulating compounds. This method of treatment is referred to ascombination therapy.

In general, compounds and methods described herein can be used togenerate hair cell growth in the ear and/or to increase the number ofhair cells in the ear (e.g., in the inner, middle, and/or outer ear).For example, the number of hair cells in the ear can be increased about2-, 3-, 4-, 6-, 8-, or 10-fold, or more, as compared to the number ofhair cells before treatment. This new hair cell growth can effectivelyrestore or establish at least a partial improvement in the subject'sability to hear. For example, administration of an agent can improvehearing loss by about 5, 10, 15, 20, 40, 60, 80, 100% or more.

Where appropriate, following treatment, the human can be tested for animprovement in hearing or in other symptoms related to inner eardisorders. Methods for measuring hearing are well-known and include puretone audiometry, air conduction, and bone conduction tests. These examsmeasure the limits of loudness (intensity) and pitch (frequency) that ahuman can hear. Hearing tests in humans include behavioral observationaudiometry (for infants to seven months), visual reinforcementorientation audiometry (for children 7 months to 3 years) and playaudiometry for children older than 3 years. Oto-acoustic emissiontesting can be used to test the functioning of the cochlea hair cells,and electro-cochleography provides information about the functioning ofthe cochlea and the first part of the nerve pathway to the brain. Insome embodiments, treatment can be continued with or withoutmodification or can be stopped.

Routes of Administration

Direct Therapy

The route of administration will vary depending on the disease beingtreated. Hair cell loss, sensorineural hearing loss, and vestibulardisorders can be treated using direct therapy using systemicadministration and/or local administration. In some embodiments, theroute of administration can be determined by a subject's health careprovider or clinician, for example following an evaluation of thesubject. In some embodiments, a individual subject's therapy may becustomized, e.g., one or more β-catenin modulating compounds, the routesof administration, and the frequency of administration can bepersonalized. Alternatively, therapy may be performed using a standardcourse of treatment, e.g., using one or more pre-selected β-cateninmodulating compounds and pre-selected routes of administration andfrequency of administration.

In some embodiments, one or more β-catenin modulating compounds can beadministered to a subject, e.g., a subject identified as being in needof treatment for hair cell loss, using a systemic route ofadministration. Systemic routes of administration can include, but arenot limited to, parenteral routes of administration, e.g., intravenousinjection, intramuscular injection, and intraperitoneal injection;enteral routes of administration e.g., administration by the oral route,lozenges, compressed tablets, pills, tablets, capsules, drops (e.g., eardrops), syrups, suspensions and emulsions; rectal administration, e.g.,a rectal suppository or enema; a vaginal suppository; a urethralsuppository; transdermal routes of administration; and inhalation (e.g.,nasal sprays).

Alternatively or in addition, one or more β-catenin modulating compoundscan be administered to a subject, e.g., a subject identified as being inneed of treatment for hair cell loss, using a local route ofadministration. Such local routes of administration includeadministering one or more compounds into the ear of a subject and/or theinner ear of a subject, for example, by injection and/or using a pump.

In some embodiments, one or more β-catenin modulating compounds can beinjected into the ear (e.g., auricular administration), such as into theluminae of the cochlea (e.g., the Scala media, Sc vestibulae, and Sctympani). For example, one or more β-catenin modulating compounds can beadministered by intratympanic injection (e.g., into the middle ear),and/or injections into the outer, middle, and/or inner ear. Such methodsare routinely used in the art, for example, for the administration ofsteroids and antibiotics into human ears. Injection can be, for example,through the round window of the ear or through the cochlea capsule.

In another mode of administration, one or more β-catenin modulatingcompounds can be administered in situ, via a catheter or pump. Acatheter or pump can, for example, direct a pharmaceutical compositioninto the cochlea luminae or the round window of the ear. Exemplary drugdelivery apparatus and methods suitable for administering one or morecompounds into an ear, e.g., a human ear, are described by McKenna etal., (U.S. Publication No. 2006/0030837) and Jacobsen et al., (U.S. Pat.No. 7,206,639). In some embodiments, a catheter or pump can bepositioned, e.g., in the ear (e.g., the outer, middle, and/or inner ear)of a subject during a surgical procedure. In some embodiments, acatheter or pump can be positioned, e.g., in the ear (e.g., the outer,middle, and/or inner ear) of a subject without the need for a surgicalprocedure.

Alternatively or in addition, one or more compounds can be administeredin combination with a mechanical device such as a cochlea implant or ahearing aid, which is worn in the outer ear. An exemplary cochleaimplant that is suitable for use with the present invention is describedby Edge et al., (U.S. Publication No. 2007/0093878).

In some embodiments, the modes of administration described above may becombined in any order and can be simultaneous or interspersed.

Alternatively or in addition, the present invention may be administeredaccording to any of the Food and Drug Administration approved methods,for example, as described in CDER Data Standards Manual, version number004 (which is available at fda.give/cder/dsm/DRG/drg00301.htm).

β-catenin Expression Constructs

In some aspects, β-catenin can be expressed using expression constructs,e.g., naked DNA constructs, DNA vector based constructs, and/or viralvector and/or viral based constructs.

The present application also provides such expression constructsformulated as a pharmaceutical composition, e.g., for administration toa subject. Such pharmaceutical compositions are not limited to oneexpression construct and rather can include two or more expressionconstructs (e.g., two, three, four, five, six, seven, eight, nine, tenor more expression constructs).

Naked DNA constructs and the therapeutic use of such constructs are wellknown to those of skill in the art (see, e.g., Chiarella et al., RecentPatents Anti-Infect. Drug Disc.,3:93-101, 2008; Gray et al., ExpertOpin. Biol. Ther., 8:911-922, 2008; Melman et al., Hum. Gene Ther.,17:1165-1176, 2008). Typically, naked DNA constructs include one or moretherapeutic nucleic acids (e.g., DNA encoding β-catenin) and a promotersequence. A naked DNA construct can be a DNA vector, commonly referredto as pDNA. Naked DNA typically do not incorporate into chromosomal DNA.Generally, naked DNA constructs do not require, or are not used inconjunction with, the presence of lipids, polymers, or viral proteins.Such constructs may also include one or more of the non-therapeuticcomponents described herein.

DNA vectors are known in the art and typically are circular doublestranded DNA molecules. DNA vectors usually range in size from three tofive kilo-base pairs (e.g., including inserted therapeutic nucleicacids). Like naked DNA, DNA vectors can be used to deliver and expressone or more therapeutic proteins in target cells. DNA vectors do notincorporate into chromosomal DNA.

Generally, DNA vectors include at least one promoter sequence thatallows for replication in a target cell. Uptake of a DNA vector may befacilitated (e.g., improved) by combining the DNA vector with, forexample, a cationic lipid, and forming a DNA complex.

Also useful are viral vectors, which are also well known to those ofskill in the art. Typically, viral vectors are double stranded circularDNA molecules that are derived from a virus. Viral vectors are typicallylarger in size than naked DNA and DNA vector constructs and have agreater capacity for the introduction of foreign (i.e., not virallyencoded) genes. Like naked DNA and DNA vectors, viral vectors can beused to deliver and express one or more therapeutic nucleic acids intarget cells. Unlike naked DNA and DNA vectors, certain viral vectorsstably incorporate themselves into chromosomal DNA.

Typically, viral vectors include at least one promoter sequence thatallows for replication of one or more vector encoded nucleic acids,e.g., a therapeutic nucleic acid, in a host cell. Viral vectors mayoptionally include one or more non-therapeutic components describedherein. Advantageously, uptake of a viral vector into a target cell doesnot require additional components, e.g., cationic lipids. Rather, viralvectors transfect or infect cells directly upon contact with a targetcell.

The approaches described herein include the use of retroviral vectors,adenovirus-derived vectors, and/or adeno-associated viral vectors asrecombinant gene delivery systems for the transfer of exogenous genes invivo, particularly into humans. Protocols for producing recombinantretroviruses and for infecting cells in vitro or in vivo with suchviruses can be found in Current Protocols in Molecular Biology, Ausubel,F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections9.10-9.14, and other standard laboratory manuals.

The genome of an adenovirus can be manipulated such that it encodes andexpresses a gene product of interest but is inactivated in terms of itsability to replicate in a normal lytic viral life cycle. See, forexample, Berkner et al., BioTechniques 6:616, 1988; Rosenfeld et al.,Science 252:431-434, 1991; and Rosenfeld et al. Cell 68:143-155, 1992.Suitable adenoviral vectors derived from the adenovirus strain Ad type 5d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) areknown to those skilled in the art. Recombinant adenoviruses can beadvantageous in certain circumstances in that they are not capable ofinfecting nondividing cells and can be used to infect a wide variety ofcell types, including epithelial cells (Rosenfeld et al. (1992) citedsupra). Furthermore, the virus particle is relatively stable andamenable to purification and concentration, and as above, can bemodified so as to affect the spectrum of infectivity. Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situ where introduced DNA becomes integrated into thehost genome (e.g., retroviral DNA). Moreover, the carrying capacity ofthe adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Berkner et al. cited supra;Haj-Ahmand and Graham, J. Virol., 57:267, 1986).

Adeno-associated virus is a naturally occurring defective virus thatrequires another virus, such as an adenovirus or a herpes virus, as ahelper virus for efficient replication and a productive life cycle. (Fora review see Muzyczka et al., Curr. Topics in Micro. and Immunol.158:97-129, 1992). It is also one of the few viruses that may integrateits DNA into non-dividing cells, and exhibits a high frequency of stableintegration (see for example Flotte et al., Am. J. Respir. Cell. Mol.Biol. 7:349-356, 1992; Samulski et al., J. Virol., 63:3822-3828, 1989;and McLaughlin et al., J. Virol., 62:1963-1973, 1989). Vectorscontaining as little as 300 base pairs of AAV can be packaged and canintegrate. Space for exogenous DNA is limited to about 4.5 kb. An AAVvector such as that described in Tratschin et al., Mol. Cell. Biol.5:3251-3260, 1985 can be used to introduce DNA into cells. Skilledpractitioners will appreciate that the use of any number of viralvectors in the presently described methods is possible.

All the molecular biological techniques required to generate anexpression construct described herein are standard techniques that willbe appreciated by one of skill in the art. Detailed methods may also befound, e.g., Current Protocols in Molecular Biology, Ausubel et al.(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 andother standard laboratory manuals. DNA encoding altered β-catenin can begenerated using, e.g., site directed mutagenesis techniques.

Polypeptides Encoding β-Catenin

Polypeptides encoding β-catenin can be generated using recombinanttechniques or using chemical synthesis. Methods for generating suchpolypeptides, and the methods required for the purification of suchpolypeptides will be appreciated by one of skill in the art.

Pharmaceutical Compositions

In some embodiments, one or more β-catenin modulating compounds can beformulated as a pharmaceutical composition. Pharmaceutical compositionscontaining one or more β-catenin modulating compounds can be formulatedaccording to the intended method of administration.

One or more β-catenin modulating compounds can be formulated aspharmaceutical compositions for direct administration to a subject.Pharmaceutical compositions containing one or more compounds can beformulated in a conventional manner using one or more physiologicallyacceptable carriers or excipients. For example, a pharmaceuticalcomposition can be formulated for local or systemic administration,e.g., administration by drops or injection into the ear, insufflation(such as into the ear), intravenous, topical, or oral administration.

The nature of the pharmaceutical compositions for administration isdependent on the mode of administration and can readily be determined byone of ordinary skill in the art. In some embodiments, thepharmaceutical composition is sterile or sterilizable. The therapeuticcompositions featured in the invention can contain carriers orexcipients, many of which are known to skilled artisans. Excipients thatcan be used include buffers (for example, citrate buffer, phosphatebuffer, acetate buffer, and bicarbonate buffer), amino acids, urea,alcohols, ascorbic acid, phospholipids, polypeptides (for example, serumalbumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, water,and glycerol. The nucleic acids, polypeptides, small molecules, andother modulatory compounds featured in the invention can be administeredby any standard route of administration. For example, administration canbe parenteral, intravenous, subcutaneous, or oral.

A pharmaceutical composition can be formulated in various ways,according to the corresponding route of administration. For example,liquid solutions can be made for administration by drops into the ear,for injection, or for ingestion; gels or powders can be made foringestion or topical application. Methods for making such formulationsare well known and can be found in, for example, Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990.

One or more β-catenin modulating compounds can be administered, e.g., asa pharmaceutical composition, directly and/or locally by injection orthrough surgical placement, e.g., to the inner ear. The amount of thepharmaceutical composition may be described as the effective amount orthe amount of a cell-based composition may be described as atherapeutically effective amount. Where application over a period oftime is advisable or desirable, the compositions of the invention can beplaced in sustained released formulations or implantable devices (e.g.,a pump).

Alternatively or in addition, the pharmaceutical compositions can beformulated for systemic parenteral administration by injection, forexample, by bolus injection or continuous infusion. Such formulationscan be presented in unit dosage form, for example, in ampoules or inmulti-dose containers, with an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, for example, sterile pyrogen-free water, before use.

In addition to the formulations described previously, the compositionscan also be formulated as a depot preparation. Such long actingformulations can be administered by implantation (e.g., subcutaneously).Thus, for example, the compositions can be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions formulated for systemic oral administrationcan take the form of tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents (forexample, pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (for example, lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(for example, magnesium stearate, talc or silica); disintegrants (forexample, potato starch or sodium starch glycolate); or wetting agents(for example, sodium lauryl sulphate). The tablets can be coated bymethods well known in the art. Liquid preparations for oraladministration may take the form of, for example, solutions, syrups orsuspensions, or they may be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations may be prepared by conventional means with pharmaceuticallyacceptable additives such as suspending agents (for example, sorbitolsyrup, cellulose derivatives or hydrogenated edible fats); emulsifyingagents (for example, lecithin or acacia); non-aqueous vehicles (forexample, almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (for example, methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations may alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration may be suitablyformulated to give controlled release of the active compound.

In some embodiments, the pharmaceutical compositions described hereincan include one or more of the compounds formulated according to any ofthe methods described above, and one or more cells obtained to themethods described herein.

Cell Therapy

In general, the cell therapy methods described herein can be used topromote complete or partial differentiation of a cell to or towards amature cell type of the inner ear (e.g., a hair cell) in vitro. Cellsresulting from such methods can be transplanted or implanted into asubject in need of such treatment. The cell culture methods required topractice these methods, including methods for identifying and selectingsuitable cell types, methods for promoting complete or partialdifferentiation of selected cells, methods for identifying complete orpartially differentiated cell types, and methods for implanting completeor partially differentiated cells are described below.

Cell Selection

Target cells suitable for use in the present invention include, but arenot limited to, cells that are capable of differentiating completely orpartially into a mature cell of the inner ear, e.g., a hair cell (e.g.,an inner ear and/or outer ear hair cell), when contacted, e.g., invitro, with one or more β-catenin modulating compounds. Exemplary cellsthat are capable of differentiating into a hair cell include, but arenot limited to stem cells (e.g., inner ear stem cells, adult stem cells,bone marrow derived stem cells, embryonic stem cells, mesenchymal stemcells, skin stem cells, and fat derived stem cells), progenitor cells(e.g., inner ear progenitor cells), support cells (e.g., Deiters' cells,pillar cells, inner phalangeal cells, tectal cells and Hensen's cells),and/or germ cells. The use of stem cells for the replacement of innerear sensory cells is described, e.g., in Li et al., (U.S. PublicationNo. 2005/0287127) and Li et al., (U.S. patent Ser. No. 11/953,797). Theuse of bone marrow derived stem cells for the replacement of inner earsensory cells is described, e.g., in Edge et al., PCT/US2007/084654.

Such suitable cells can be identified by analyzing (e.g., qualitativelyor quantitatively) the presence of one or more tissue specific genes.For example, gene expression can be detected by detecting the proteinproduct of one or more tissue-specific genes. Protein detectiontechniques involve staining proteins (e.g., using cell extracts or wholecells) using antibodies against the appropriate antigen. In this case,the appropriate antigen is the protein product of the tissue-specificgene expression. Although, in principle, a first antibody (i.e., theantibody that binds the antigen) can be labeled, it is more common (andimproves visualization) to use a second antibody directed against thefirst (e.g., an anti-IgG). This second antibody is conjugated eitherwith fluorochromes, or appropriate enzymes for colorimetric reactions,or gold beads (for electron microscopy), or with the biotin-avidinsystem, so that the location of the primary antibody, and thus theantigen, can be recognized.

Tissue-specific gene expression can also be assayed by detection of RNAtranscribed from the gene. RNA detection methods include reversetranscription coupled to polymerase chain reaction (RT-PCR), Northernblot analysis, and RNAse protection assays.

Exemplary tissue specific genes that may be used to identify a stem cell(e.g., an undifferentiated cell) include, but are not limited to, e.g.,nestin, sox1, sox2, or musashi, NeuroD, Atoh1, and neurogenin1.Alternatively or in addition, stem cells can be selected based on one ormore of the unique properties that such cell types present in vitro. Forexample, in vitro, stem cells often show a distinct potential forforming spheres by proliferation of single cells. Thus, theidentification and isolation of spheres can aid in the process ofisolating stem cells from mature tissue for use in making differentiatedcells of the inner ear. For example, stem cells can be cultured in serumfree DMEM/high-glucose and F12 media (mixed 1:1), and supplemented withN2 and B27 solutions and growth factors. Growth factors such as EGF,IGF-1, and bFGF have been demonstrated to augment sphere formation inculture.

Exemplary tissue specific genes that may be used to identify aprogenitor cells and/or an inner ear progenitor cell (e.g., a less thanfully differentiated or partially differentiated cell) include but arenot limited to, e.g., nestin, sox2, and musashi, in addition to certaininner-ear specific marker genes such as Brn3c, islet1 and Pax2

Exemplary tissue specific genes that may be used to identify fullydifferentiated support cells include, but are not limited to, e.g.,p27_(kip), p75, S100A, Jagged-1, and Prox1.

Exemplary tissue specific genes that may be used to identify fullydifferentiated cells capable of functioning as inner ear sensory cells)include, but are not limited to, e.g., myosin VIIa, Math1 (Atoh1), α9acetylcholine receptor, espin, parvalbumin 3, and F-actin (phalloidin).

Alternatively or in addition, cells suspected as being fullydifferentiated (e.g., cells capable of functioning as inner ear sensorycells) may be subjected to physiological testing to determine whetherconductance channels that would be present in mature hair cells arepresent and active.

Alternatively or in addition, inner ear hair cells may be distinguishedfrom other fully differentiated cells of the inner ear (e.g., spiralganglia) by analyzing the expression of markers that are specific tospiral ganglia, which include but are not limited to ephrinB2, ephrinB3,trkB, trkC, GATA3, and BF1. In some embodiments, cells identified asexpressing one or more markers that are specific to spiral ganglia,e.g., ephrinB2, ephrinB3, trkB, trkC, GATA3, and BF1 will be isolatedand removed.

In some embodiments, suitable cells can be derived from a mammal, suchas a human, mouse, rat, pig, sheep, goat, or non-human primate. Forexample, stem cells have been identified and isolated from the mouseutricular macula (Li et al., Nature Medicine 9:1293-1299, 2003). Thecells can also be obtained from a subject to whom they will subsequentlybe readministered.

In some embodiments, target cells can be isolated from the inner ear ofan animal. More specifically, a suitable cells can be obtained from thecochlea organ of Corti, the modiolus (center) of the cochlea, the spiralganglion of the cochlea, the vestibular sensory epithelia of thesaccular macula, the utricular macula, or the cristae of thesemicircular canals.

In some embodiments, target cells can be any cell that expresses or canexpress Atoh1. In some embodiments, target cells can be obtained fromtissues such as bone marrow, blood, skin, or an eye. In someembodiments, target cells can be obtained from any tissue that expressesor can express Atoh1, for example, intestinal tissue, skin (e.g.,Merkel's cells), and cerebellum.

In some embodiments, target cells can be obtained from a single source(e.g., the ear or a structure or tissue within the ear) or a combinationof sources (e.g., the ear and one or more peripheral tissues (e.g., bonemarrow, blood, skin, or an eye)).

Alternatively or in addition, methods include obtaining tissue from theinner ear of the animal, where the tissue includes at least a portion ofthe utricular maculae. The animal can be a mammal, such as a mouse, rat,pig, rabbit, goat, horse, cow, dog, cat, primate, or human. The isolatedtissue can be suspended in a neutral buffer, such as phosphate bufferedsaline (PBS), and subsequently exposed to a tissue-digesting enzyme(e.g., trypsin, leupeptin, chymotrypsin, and the like) or a combinationof enzymes, or a mechanical (e.g., physical) force, such as trituration,to break the tissue into smaller pieces. Alternatively, or in addition,both mechanisms of tissue disruption can be used. For example, thetissue can be incubated in about 0.05% enzyme (e.g., about 0.001%,0.01%, 0.03%, 0.07%, or 1.0% of enzyme) for about 5, 10, 15, 20, or 30minutes, and following incubation, the cells can be mechanicallydisrupted. The disrupted tissue can be passed through a device, such asa filter or bore pipette, that separates a stem cell or progenitor cellfrom a differentiated cell or cellular debris. The separation of thecells can include the passage of cells through a series of filtershaving progressively smaller pore size. For example, the filter poresize can range from about 80 μm or less, about 70 μm or less, about 60μm or less, about 50 μm or less, about 40 μm or less, about 30 μm orless, about 35 μm or less, or about 20 μm or less.

The cells obtained may constitute an enriched population of stem cellsand/or progenitor cells; isolation from all (or essentially all)differentiated cells or other cellular material within the tissue may beachieved but is not required to meet the definition of “isolated.”Absolute purity is not required. The invention encompasses cellsobtained by the isolation procedures described herein. The cells may bemixed with a cryoprotectant and stored or packaged into kits. Onceobtained, the stem cells and/or progenitor cells can be expanded inculture.

Where a mixed population of cells is used, the proportion of stem cellswithin the test population can vary. For example, the population cancontain few stem cells (e.g., about 1-10%) a moderate proportion of stemcells (e.g., about 10-90% (e.g., about 20, 25, 30, 40, 50, 60, 70, 75,80, or 85% stem cells)) or many stem cells (e.g., at least 90% of thepopulation (e.g., 92, 94, 96, 97, 98, or 99%) can be stem cells). Thecells will have the potential to differentiate into a completely orpartially differentiated cell of the inner ear (e.g., the cell can be apluripotent stem cell that differentiates into a cell that expresses oneor more auditory proteins). Partially differentiated cells are useful inthe treatment methods (whether therapeutic or prophylactic) so long asthey express a sufficient number and type of auditory-specific proteinsto confer a benefit on the subject (e.g., improved hearing).

Differentiation Methods

In general, differentiation can be promoted by contacting a suitabletarget cell and/or cell population with one or more β-catenin modulatingcompounds for a time sufficient to promote complete or partialconversion (e.g., differentiation) of the target cells to or towards amature sensory cell of the inner ear, e.g., a hair cell.

Suitable target cells, e.g., identified according to the methodsdescribed above, can be cultured in vitro. In general, standard culturemethods are used in the methods described herein. Appropriate culturemedium is described in the art, such as in Li et al. Nature Medicine9:1293-1299, 2003. The growth medium for cultured stem cells can containone or more or any combination of growth factors. For example, growthmedia can contain leukemia inhibitory factor (LIF), which prevents stemcells from differentiating.

Target cells can be separated into individual well of a culture dish andcultured. Formation of spheres (clonal floating colonies) from theisolated cells can be monitored, and the spheres can be amplified bydisrupting them (e.g., by physically means) to separate the cells, andthe cells can be cultured again to form additional spheres. Suchcultured cells can then be contacted with one or more β-cateninmodulating compounds.

Alternatively or in addition, target cells may be contacted with one ormore β-catenin modulating compounds in combination with an additionalinduction protocol. There are a number of induction protocols known inthe art for inducing differentiation of stem cells with neurogenicpotential into neural progenitor cells, including growth factortreatment (e.g., treatment with EGF, FGF, and IGF, as described herein)and neurotrophin treatment (e.g., treatment with NT3 and BDNF, asdescribed herein). Other differentiation protocols are known in the art;see, e.g., Corrales et al., J. Neurobiol. 66(13):1489-500 (2006); Kim etal., Nature 418, 50-6 (2002); Lee et al., Nat Biotechnol 18, 675-9(2000); and Li et al., Nat Biotechnol., 23, 215-21 (2005).

As one example of an additional induction protocol, target cells aregrown in the presence of supplemental growth factors that inducedifferentiation into progenitor cells. These supplemental growth factorsare added to the culture medium. The type and concentration of thesupplemental growth factors is be adjusted to modulate the growthcharacteristics of the cells (e.g., to stimulate or sensitize the cellsto differentiate) and to permit the survival of the differentiated cellssuch as neurons, glial cells, supporting cells or hair cells.

Exemplary supplementary growth factors include, but are not limited tobasic fibroblast growth factor (bFGF), insulin-like growth factor (IGF),and epidermal growth factor (EGF). Alternatively, the supplementalgrowth factors can include the neurotrophic factors neurotrophin-3 (NT3)and brain derived neurotrophic factor (BDNF). Concentrations of growthfactors can range from about 100 ng/mL to about 0.5 ng/mL (e.g., fromabout 80 ng/mL to about 3 ng/mL, such as about 60 ng/mL, about 50 ng/mL,about 40 ng/mL, about 30 ng/mL, about 20 ng/mL, about 10 ng/mL, or about5 ng/mL).

Alternatively or in addition, the medium can be exchanged for mediumlacking growth factors. For example, the medium can be serum-freeDMEM/high glucose and F12 media (mixed 1:1) supplemented with N2 and B27solutions. Equivalent alternative media and nutrients can also be used.Culture conditions can be optimized using methods known in the art.

Methods for Analyzing Complete or Partial Differentiation

Target cells that have been contacted with one or more β-cateninmodulating compounds can be analyzed to determine if complete of partialdifferentiation has occurred. Such a determination can be performed byanalyzing the presence or absence of tissue specific genes, as describedabove (see Cell Selection). Alternatively or in addition, a hair cellcan be identified by physiological testing to determine if the cellsgenerate conductance channels characteristic of mature hair or spiralganglion cells. Such cells can be distinguished from spiral gangliacells using the markers described above.

Secondary assays can be used to confirm, or provide additional evidence,that a cell has differentiated into a cell of the inner ear. Forexample, a gene useful as a marker for identifying a cell of the innerear can be expressed exclusively in a particular cell type (e.g.,exclusively in a hair cell or exclusively in cells of the spiralganglion), or the cell may also be expressed in a few other cell types(preferably not more than one, two, three, four, or five other celltypes). For example, ephrinB1 and ephrinB2 are expressed in spiralganglion cells, and also in retinal cells. Thus detection of ephrinB1 orephrinB2 expression is not definitive proof that a stem cell hasdifferentiated into a cell of the spiral ganglion. Secondary assays canbe used to confirm that a cell has developed into a cell of the spiralganglion. Such assays include detection of multiple genes known to beexpressed in the suspected cell type. For example, a cell that expressesephrinB1 and/or ephrinB2, can also be assayed for expression of one ormore of GATA3, trkB, trkC, BF1, FGF10, FGF3, CSP, GFAP, and Islet1. Adetermination that these additional genes are expressed is additionalevidence that a stem cell has differentiated into a spiral ganglioncell.

Secondary assays also include detection of the absence of geneexpression or the absence of proteins that are not typically expressedin hair cells. Such negative markers include the pan-cytokeratin gene,which is not expressed in mature hair cells but is expressed insupporting cells of the inner ear (Li et al., Nature Medicine9:1293-1299, 2003).

Cells that are confirmed to have undergone complete or partialdifferentiation towards a inner ear sensory cell, e.g., a hair cell canbe transplanted or implanted into a subject.

Implantation Methods

Partially and/or fully differentiated cells, e.g., generated by themethods described above, can be transplanted or implanted, such as inthe form of a cell suspension, into the ear by injection, such as intothe luminae of the cochlea. Injection can be, for example, through theround window of the ear or through the bony capsule surrounding thecochlea. The cells can be injected through the round window into theauditory nerve trunk in the internal auditory meatus or into the scalatympani.

To improve the ability of transplanted or implanted cells to engraft,cells can be modified prior to differentiation. For example, the cellscan be engineered to overexpress one or more anti-apoptotic genes in theprogenitor or differentiated cells. The Fak tyrosine kinase or Akt genesare candidate anti-apoptotic genes that can be useful for this purpose;overexpression of FAK or Akt can prevent cell death in spiral ganglioncells and encourage engraftment when transplanted into another tissue,such as an explanted organ of Corti (see for example, Mangi et al., Nat.Med. 9:1195-201, 2003). Neural progenitor cells overexpressing α_(v)β₃integrin may have an enhanced ability to extend neurites into a tissueexplant, as the integrin has been shown to mediate neurite extensionfrom spiral ganglion neurons on laminin substrates (Aletsee et al.,Audiol. Neurootol. 6:57-65, 2001). In another example, ephrinB2 andephrinB3 expression can be altered, such as by silencing with RNAi oroverexpression with an exogenously expressed cDNA, to modify EphA4signaling events. Spiral ganglion neurons have been shown to be guidedby signals from EphA4 that are mediated by cell surface expression ofephrin-B2 and -B3 (Brors et al., J. Comp. Neurol. 462:90-100, 2003).Inactivation of this guidance signal may enhance the number of neuronsthat reach their target in an adult inner ear. Exogenous factors such asthe neurotrophins BDNF and NT3, and LIF can be added to tissuetransplants to enhance the extension of neurites and their growthtowards a target tissue in vivo and in ex vivo tissue cultures. Neuriteextension of sensory neurons can be enhanced by the addition ofneurotrophins (BDNF, NT3) and LIF (Gillespie et al., NeuroReport12:275-279, 2001).

In some embodiments, the cells described herein can be used in a cochleaimplant, for example, as described in Edge et al., (U.S. Publication No.2007/0093878). A cochlea implant is an electronic device that is used toimprove hearing in humans who have experienced hearing loss,particularly severe to profound hearing loss. These devices typicallyinclude an “external” and an “internal” part. The external part includesa microphone, which can be placed behind the ear, that detects sounds inthe environment. The sounds are then digitized and processed by a smallcomputer called a speech processor. The external components may bereferred to as a processor unit. In addition to the microphone andspeech processor, the external portion of the implant can include apower source, such as a battery and an external antenna transmittercoil. The internal part is an electronic device that is put under theskin in the vicinity of the ear and is commonly referred to as astimulator/receiver unit (see FIG. 1). The coded signal output by thespeech processor is transmitted transcutaneously to the implantedstimulator/receiver unit situated within a recess of the temporal boneof the implantee. This transcutaneous transmission occurs through use ofan inductive coupling provided between the external antenna transmittercoil which is positioned to communicate with the implanted antennareceiver coil provided with the stimulator/receiver unit. Thecommunication is typically provided by a radio frequency (RF) link, butother such links have been proposed and implemented with varying degreesof success.

The implanted stimulator/receiver unit typically includes the antennareceiver coil that receives the coded signal and power from the externalprocessor component, and a stimulator that processes the coded signaland outputs a stimulation signal to an electrode assembly, which appliesthe electrical stimulation directly to the auditory nerve producing ahearing sensation corresponding to the original detected sound.

An electrode connected to the electronic device is inserted into theinner ear. The electrode can be a bundle of wires that have opencontacts spread along the length of the cochlea and represent differentfrequencies of sounds. The number of electrodes can vary from 1 to about30 electrodes, such as about 5, 10, 15, 18, 20, 22, 24, 26, or 28electrodes.

Combination Therapies

In some embodiments, the present invention provides methods for treatinga subject with one or more compounds using the direct administration andcell therapy methods described above.

Effective Dose

Toxicity and therapeutic efficacy of the compounds and pharmaceuticalcompositions described herein can be determined by standardpharmaceutical procedures, using either cells in culture or experimentalanimals to determine the LD₅₀ (the dose lethal to 50% of the population)and the ED₅₀ (the dose therapeutically effective in 50% of thepopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index and can be expressed as the ratio LD₅₀/ED₅₀.Polypeptides or other compounds that exhibit large therapeutic indicesare preferred.

Data obtained from cell culture assays and further animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity, andwith little or no adverse effect on a human's ability to hear. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the methods described herein, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (that is, the concentrationof the test compound which achieves a half-maximal inhibition ofsymptoms) as determined in cell culture. Such information can be used tomore accurately determine useful doses in humans. Exemplary dosageamounts of a differentiation agent are at least from about 0.01 to 3000mg per day, e.g., at least about 0.00001, 0.0001, 0.001, 0.01, 0.1, 1,2, 5, 10, 25, 50, 100, 200, 500, 1000, 2000, or 3000 mg per kg per day,or more.

The formulations and routes of administration can be tailored to thedisease or disorder being treated, and for the specific human beingtreated. A subject can receive a dose of the agent once or twice or moredaily for one week, one month, six months, one year, or more. Thetreatment can continue indefinitely, such as throughout the lifetime ofthe human. Treatment can be administered at regular or irregularintervals (once every other day or twice per week), and the dosage andtiming of the administration can be adjusted throughout the course ofthe treatment. The dosage can remain constant over the course of thetreatment regimen, or it can be decreased or increased over the courseof the treatment.

Generally the dosage facilitates an intended purpose for bothprophylaxis and treatment without undesirable side effects, such astoxicity, irritation or allergic response. Although individual needs mayvary, the determination of optimal ranges for effective amounts offormulations is within the skill of the art. Human doses can readily beextrapolated from animal studies (Katocs et al., Chapter 27 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990). Generally, the dosage required toprovide an effective amount of a formulation, which can be adjusted byone skilled in the art, will vary depending on several factors,including the age, health, physical condition, weight, type and extentof the disease or disorder of the recipient, frequency of treatment, thenature of concurrent therapy, if required, and the nature and scope ofthe desired effect(s) (Nies et al., Chapter 3, In: Goodman & Gilman's“The Pharmacological Basis of Therapeutics”, 9th Ed., Hardman et al.,eds., McGraw-Hill, New York, N.Y., 1996).

Methods of Screening

In some embodiments, a candidate compound can be tested for its abilityto increase β-catenin levels (e.g., protein levels) and/or activity(e.g., biological activity) in target cells and/or to promote anincrease in the levels (e.g. protein levels) and/or activity (e.g.,biological activity) of β-catenin in the nucleus of target cells usingcells (e.g., stem cells) that have been engineered to express aβ-catenin reporter construct. These engineered cells make up a reportercell line. A reporter construct includes (1) any gene or nucleic acidsequence whose expression may be indirectly or directly detected and/orassayed; and (2) a β-catenin reporter sequence (e.g., any nucleic acidsequence whose expression is specifically correlated with β-cateninactivity or expression), wherein (2) is operably linked to (1) such that(2) drives the expression of (1). S Examples of (1) include, withoutlimitation, green fluorescent protein (GFP), α-glucuronidase (GUS),luciferase, chloramphenicol transacetylase (CAT), horseradish peroxidase(HRP), alkaline phosphatase, acetylcholinesterase and (3-galactosidase.Other optional fluorescent reporter genes include but are not limited tored fluorescent protein (RFP), cyan fluorescent protein (CFP) and bluefluorescent protein (BFP), or any paired combination thereof, providedthe paired proteins fluoresce at distinguishable wavelengths. Examplesof a β-catenin reporter sequence include β-catenin transcriptionalbinding sequences (e.g., nucleic acid sequences that can be bound (e.g.,specifically bound) by β-catenin, wherein binding of β-catenin to thesequence modulates expression of the sequence (e.g., a promoter sequencethat can be bound by β-catenin)). In some embodiments, a candidatecompound can be assessed using the TOPflash genetic reporter system(Chemicon).

Alternatively or in addition, a reporter gene can be under control of apromoter that is active in cells of the inner ear, including progenitorcells and cells at varying degrees of differentiation, but not in stemcells. In such cases, ideally, the promoter is stably upregulated in thedifferentiated cells or progenitors cells to allow assessment of thepartially or fully differentiated phenotype (e.g., expression of thereporter gene and further identification of genes known to be expressedin the inner ear).

Methods for Assessing β-Catenin Levels and/or Activity

β-catenin levels (e.g., protein levels) and/or activity (e.g.,biological activity) in target cells and/or in the nucleus of targetcells can be assessed using standard methods such as Western Blotting,reverse transcriptase polymerase chain reaction, immunocytochemistry,and genetic reporter assays, examples of each of which are providedherein. Increases in β-catenin levels (e.g., protein levels) and/oractivity (e.g., biological activity) in target cells and/or in thenucleus of target cells can be assessed by comparing β-catenin levelsand/or activity in a first sample or a standard with β-catenin levelsand/or activity in a second sample, e.g., after treatment of the sampleusing a method or composition expected to increase β-catenin levelsand/or activity.

Kits

The compounds and pharmaceutical compositions described herein can beprovided in a kit, as can cells that have been induced to differentiate(e.g., stem cells, progenitor cells, and/or support cells that havedifferentiated into, for example, hair cells or hair-like cells) and/orthat are capable of differentiating into hair cells. The kit can alsoinclude combinations of the compounds, pharmaceutical compositions, andcells described herein. The kit can include (a) one or more compounds,such as in a composition that includes the compound, (b) cells that havebeen induced to differentiate (e.g., stem cells, progenitor cells,and/or support cells that have differentiated into, for example, haircells or hair-like cells) and/or that are capable of differentiatinginto hair cells, (c) informational material, and any combination of(a)-(c). The informational material can be descriptive, instructional,marketing or other material that relates to the methods described hereinand/or to the use of the agent for the methods described herein. Forexample, the informational material relates to the use of the compoundto treat a subject who has, or who is at risk for developing, a auditoryhair cell loss hearing. The kits can also include paraphernalia foradministering one or more compounds to a cell (in culture or in vivo)and/or for administering a cell to a patient, and any combination of themethods described herein.

In one embodiment, the informational material can include instructionsfor administering the pharmaceutical composition and/or cell(s) in asuitable manner to treat a human, e.g., in a suitable dose, dosage form,or mode of administration (e.g., a dose, dosage form, or mode ofadministration described herein). In another embodiment, theinformational material can include instructions to administer thepharmaceutical composition to a suitable subject, e.g., a human, e.g., ahuman having, or at risk for developing, auditory hair cell loss.

The informational material of the kits is not limited in its form. Inmany cases, the informational material (e.g., instructions) is providedin printed matter, such as in a printed text, drawing, and/orphotograph, such as a label or printed sheet. However, the informationalmaterial can also be provided in other formats, such as Braille,computer readable material, video recording, or audio recording. Ofcourse, the informational material can also be provided in anycombination of formats.

In addition to the compound, the composition of the kit can includeother ingredients, such as a solvent or buffer, a stabilizer, apreservative, a fragrance or other cosmetic ingredient, and/or a secondagent for treating a condition or disorder described herein.Alternatively, the other ingredients can be included in the kit, but indifferent compositions or containers than the compound. In suchembodiments, the kit can include instructions for admixing the agent andthe other ingredients, or for using one or more compounds together withthe other ingredients.

The kit can include one or more containers for the pharmaceuticalcomposition. In some embodiments, the kit contains separate containers,dividers or compartments for the composition and informational material.For example, the composition can be contained in a bottle (e.g., adropper bottle, such as for administering drops into the ear), vial, orsyringe, and the informational material can be contained in a plasticsleeve or packet. In other embodiments, the separate elements of the kitare contained within a single, undivided container. For example, thecomposition is contained in a bottle, vial or syringe that has attachedthereto the informational material in the form of a label. In someembodiments, the kit includes a plurality (e.g., a pack) of individualcontainers, each containing one or more unit dosage forms (e.g., adosage form described herein) of the pharmaceutical composition. Forexample, the kit can include a plurality of syringes, ampoules, foilpackets, or blister packs, each containing a single unit dose of thepharmaceutical composition. The containers of the kits can be air tightand/or waterproof, and the containers can be labeled for a particularuse. For example, a container can be labeled for use to treat a hearingdisorder.

As noted above, the kits optionally include a device suitable foradministration of the composition (e.g., a syringe, pipette, forceps,dropper (e.g., ear dropper), swab (e.g., a cotton swab or wooden swab),or any such delivery device).

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

An adenoviral library was employed to test the affect of a number ofgene on Atoh1 expression. Preliminary data generated using this methodindicated that β-catenin modulated the expression of Atoh1. To confirmand characterize these findings, β-catenin was expressed in varioushuman and non-human cell lines and animal models as described in thesubsequent Examples.

Example 1 β-Catenin Modulates Atoh1 mRNA Expression in Human Cells

Human embryonic kidney (HEK) cells and the human intestinal epithelialcell line HT29 (Human colon adenocarcinoma grade II cell line) weremaintained in culture media containing Dulbecco's Modified Eagles Medium(DMEM) supplemented with 10% heat inactivated fetal calf serum (FCS), 2mM Glutamax, penicillin (50 U/mL), and streptomycin (50 μg/mL) usingstandard cell culture methods. For β-catenin overexpression experiments,10⁶ HEK and HT29 cells were seeding per 10 cm dish.

β-catenin overexpression was achieved by transfecting HEK and HT29 cellsseeded as described above with 5 μg of pcDNA3 (Invitrogen) encodinghuman β-catenin under the control of a cytomegalovirus (CMV) promoter(Michiels et al., Nature Biotechnology, 20:1154-1157, 2002). Negativecontrol cells included untransfected cells and cells transfected with 5μg green fluorescent protein, under the control of a CMV promoter (GFP:Michiels et al., supra). Positive control cells were transfected with 5μg of Atoh1 under the control of a CMV promoter (Lumpkin et al., GeneExpr. Patterns, 3:389-395, 2003). All transfections were performed using15 μL Lipofectamine™ 2000 for four hours, according to themanufacturer's instruction (Invitrogen). At the four hour time point,the transfection solution was replaced with culture media. Cells werethen cultured for a total of 24 hours before RNA extractions wereperformed using the RNeasy Mini kit, according to the manufacturer'sinstruction (Qiagen). 1 μg RNA was then subjected to reversetranscriptase polymerase chain reaction using SuperTranscript™ III andTaq DNA polymerase, according to the manufacturer's instruction (NewEngland Biolabs), using the following primer pairs:

Atoh1 (human):  (SEQ ID NO: 2) Sense: 5′-GCGCAATGTTATCCCGTCGTT-3′(SEQ ID NO: 3) Antisense: 5′-AAAATTCCCCGTCGCTTCTGTG-3′Glyceraldehyde 3-phosphate dehydrogenase (GAPDH-human) (SEQ ID NO: 4)Sense: 5′-CTTTTAACTCTGGTAAAGTGG-3′ (SEQ ID NO: 5) Antisense:5′-TTTTGGCTCCCCCCTGCAAAT-3′

Annealing temperatures and cycles were optimized for each primer pair.The polymerase chain reaction (PCR) products that resulted from theabove Atoh1 and GAPDH primer pairs were 479 base pairs (bp) and 287 bp,respectively. PCR products were resolved and analyzed by agarose gelelectrophoresis.

As shown in FIGS. 1A and 1B, β-catenin expression promoted an increasein Atoh1 mRNA expression in HEK and HT29, respectively, which wassimilar to the increase promoted by cells transfected with Atoh1 as apositive control in each cell line. In contrast, untransfected and GFPtransfected cells did not show an increase in Atoh1 mRNA expression.

The Atoh1 upregulation observed in FIG. 1 was quantified in HEK cellsusing real-time PCR (RT-PCR). Briefly, cells were cultured andtransfected as described above. RT-PCR primers Atoh1 and S18 werepurchased from Applied Biosystems and RT-PCR was performed using aPerkin Elmer ABI PRISM™ 7700 Sequence Detector (PE Applied Biosystems).Two independent experiments were performed in triplicate and Atoh1expression was expressed as the mean value relative to the expression ofthe housekeeping gene, S18.

As shown in FIG. 1B, Atoh1 expression increased in HEK cells 36.02±4.46fold compared to untreated control cells.

Similar experiments were also performed using neural progenitor cells.

Neural progenitor cells were obtained using ROSA26 mouse embryonic stemcells (Zambrowicz et al., Proc. Natl. Acad. Sci. USA., 94:3789-3794,1997) using the methods described in Li et al. (BMC Neurosci., 10:122,2009). β-catenin was overexpressed as described above.

Atoh1 and β-catenin levels were determined following β-cateninoverexpression by subjecting 1 μg of RNA to RT-PCR usingSuperTranscript™ III and Taq DNA polymerase (New England Biolabs), asdescribed above. GAPDH levels were assessed as control. Levels of eachof the markers were assessed using the following oligonucleotideprimers:

Atoh1:  (SEQ ID NO: 6) Sense: 5′-AGATCTACATCAACGCTCTGTC′-3′(SEQ ID NO: 7) Antisense: 5′-ACTGGCCTCATCAGAGTCACTG-3′ β-catenin:(SEQ ID NO: 8) Sense: 5′-ATGCGCTCCCCTCAGATGGTGTC-3′ (SEQ ID NO: 9)Antisense: 5′-TCGCGGTGGTGAGAAAGGTTGTGC-3′ GAPDH: (SEQ ID NO: 10) Sense:5′-AACGGGAAGCCCATCACC-3′ (SEQ ID NO: 11) Antisense:5′-TCGCGGTGGTGAGAAAGGTTGTGC-3′

As shown in FIGS. 1C and 1D, Atoh1 mRNA expression was upregulated inneural progenitor cells following β-catenin expression (741.2±218.2)compared to untransfected control cells or cells transfected with GFP(1±0.2). As expected, Atoh1 expression also increased followingtransfection with Atoh1.

These observations suggest that β-catenin increases Atoh1 mRNAexpression in human cell lines.

Example 2 β-Catenin Modulates Atoh1 Protein Expression in Human Cells

Atoh1 protein expression was analyzed in HEK cells transfected asdescribed in Example 1. Following transfection, cells were cultured for72 hours. Proteins were then resolved on 4-12% nuPAGE® Bis-Tris gels(Invitrogen) and transferred to 0.2 μm nitrocellulose membranes(BioRad). Membranes were then immunoblotted with mouse anti-Atoh1antibody (Developmental Studies Hybridoma bank) followed byHRP-conjugated anti-mouse antibody (Sigma). Immunoblots were processedusing ECL™, according to the manufacturer instructions (AmershamPharmacia).

As shown in FIG. 3, Atoh1 was not detectable in untransfected controlHEK cells or GFP transfected cells. In contrast, Atoh1 was detectable inHEK cells transfected with β-catenin and Atoh1.

These observations suggest that β-catenin increases Atoh1 proteinexpression in human cell lines. Atoh1 expression also increasedfollowing transfection with Atoh1 possibly due to the activation ofendogenous Atoh1 via an Atoh1 auto-feedback loop (Helms et al.,Development, 127:1185-1196, 2000).

Example 3 β-Catenin Modulates Atoh1 mRNA Expression in Mouse Cells

Murine Neuro2a cells and mouse neural progenitor cells derived frommouse ES cells (mES) were cultured and transfected as described inExample 1. Atoh1 and GAPDH mRNA was amplified using PCR and thefollowing primer pairs:

Atoh1 (mouse) (SEQ ID NO: 12) Sense: 5′-GCGCAATGTTATCCCGTCGTT-3′(SEQ ID NO: 13) Antisense: 5′-AAAATTCCCCGTCGCTTCTGTG-3′ GAPDH (mouse)(SEQ ID NO: 14) Sense: 5′-CTTTTAACTCTGGTAAAGTGG-3′ (SEQ ID NO: 15)Antisense: 5′-TTTTGGCTCCCCCCTGCAAAT-3′

Annealing temperatures and cycles were optimized for each primer pair.The polymerase chain reaction (PCR) products that resulted from theabove Atoh1 and GAPDH primer pairs were 479 base pairs (bp) and 287 bp,respectively. PCR products were resolved and analyzed by agarose gelelectrophoresis.

As shown in FIGS. 4A and 4B, β-catenin expression promoted an increasein Atoh1 mRNA expression in Neuro2a and mES cells, respectively. Thisincrease was similar to the increase promoted by cells transfected withAtoh1 as a positive control in both cell lines. In contrast,untransfected and GFP transfected cells did not show an increase inAtoh1 mRNA expression.

The Atoh1 upregulation observed in FIG. 4 was quantified in Neuro2acells using real-time PCR (RT-PCR), as described in Example 1.

As shown in FIG. 5, Atoh1 expression increased in Neuro2a cells871.86±141.31 fold compared to untreated control cells.

Neuro2a data is also shown in FIGS. 1C and 1D.

These observations suggest that β-catenin increases Atoh1 mRNAexpression in murine cell lines.

The data shown in Examples 1-3 was corroborated using gene silencing.Briefly, siRNA were designed to silence Atoh1 (NM_007500.4, NM_005172.1)and β-catenin (NM_007614.2 and NM_001904.3), as shown below:

Atoh1: (SEQ ID NO: 16) GCAACGUUAUCCCGUCCUUUAACAGCGAUGAUGGCACAβ-catenin:  (SEQ ID NO: 17) GCGCUUGGCUGAACCAUCAUUGUGAAAUUCUUGGCUAUUAUU

200 nM of each siRNA were transfected using the GeneSilencer™transfection reagent at 5 μL/mL. Cells were incubated in the presence ofthe transfection mix for 16 hours and were harvested following a totalof 48 hours. Non-targeting siRNA was used as control. Gene silencing wasconfirmed using RT-PCR. Cells were also transfected with Atoh1,β-catenin, or GFP using the methods described above.

As shown in FIGS. 1E and F, Atoh1 expression was decreased by siRNAdirected against Atoh1 and β-catenin in both neural progenitor cells(see Example 2) and Neuro2a (Atoh1 expression in the presence of Atoh1siRNA decreased by about 45%. Atoh1 expression in the presenceofβ-catenin decreased by about 40%). β-catenin also suppressed β-cateninexpression levels in all cell types tested.

The correlation between β-catenin and Atoh1 was also corroborated usinggenetic reporter assays. Briefly, 10⁵ Neuro2a cells were seeded into a24-well plate one day prior to transfection. 0.125 μg ofAtoh1-luciferase reporter construct and 0.125 μg o CBFI-luciferasereporter construct (Hseih et al., Mol. Cell. Biol., 16(3):952-959, 1996)TOPFlash or FOPFlas (Addgene) or 0.125 μg of Renilla-luciferase weremixed in the presence or absence of 0.25 μg β-catenin and 0.5 μLlipofectamine 2000 in 0.125 mL of opti-MEM. This transfection mixturewas then incubated on the cells for 4 hours. Cells were lysed after 48hours and luciferase activity was measured using the Dual LuciferaseReporter Assay System (Promega) in a TD-20/20 Luminometer (TurnerDesigns).

As shown in FIGS. 1G and H, reporter activity of TOPFlash (whichcontains multiple β-catenin binding sites) was comparable to reporteractivity of the Atoh1 reporter, indicating that Atoh1 is regulated byβ-catenin (FIG. 1G). Increased β-catenin expression also raised thelevel of the active fraction of nuclear β-catenin as detected using anantibody that binds specifically to the unphosphorylated form (FIG. 1H)(unphosphorylated β-catenin was detected using the anti-unphosphorylated13-catenin antibody disclosed by van Noort et al., Blood,110(7):2778-2779, 2007).

Nuclear unphosphorylatedβ-catenin and Atoh1 levels were also increasedwhen cells were incubated in Wnt3a conditioned media (FIG. 1H). Incontrast, overexpression of dominant negative Tcf4, which lacks theβ-catenin binding site, decreased the level of Atoh1.

Example 4 β-Catenin Directly Interacts with the Atoh1 Enhancer Region

To investigate whether β-catenin, in combination with Tcf-Lef factor,has a direct interaction with regulatory regions of the Atoh1 gene, DNAbinding to β-catenin was analyzed using chromatin immunoprecipitation(ChIP).

10⁷ HEK cells were crosslinked in DMEM containing 1% formaldehyde for 10minutes followed by 5 minutes at 37° C. in formaldehyde saturated with0.125 M glycine. Crosslinked cells were harvested, rinsed in phosphatebuffered saline (PBS), and centrifuged for 5 minutes at 160 g at 4° C.in cold PBS. Samples were then resuspended in sonication buffer (1%Triton® X-100, 0.1% deoxycholate, 50 mM Tris pH 8.1, 150 mM NaCl, 5 mMethylenediaminetetraacetic acid (EDTA), 2 mMphenylmethanesulphonylfluoride (PMSF), and a 1:100 dilution of freshproteinase inhibitor cocktail (Sigma)) and genomic DNA was sheared using15 pulses (5 seconds/pulse) in a sonication bath. Cell extracts werepelleted and resuspended in 1 ml radioimmunoprecipitation assay (RIPA)buffer supplemented with fresh proteinase inhibitors (Sigma). Eachsample was then separated into one 200 μL aliquot and two 400 μLaliquots. The 200 μL aliquot was not subjected to immunoprecipitation,but was used as the input control for the subsequent PCR reaction(input). The first 400 μL aliquot was immunoprecipitated using mouseanti-β-catenin antibody (Upstate, 05-601) as the primary antibody at adilution of 1:100. The second 400 μL aliquot was immunoprecipitatedusing nonimmune IgG as the primary antibody at a concentration of 1:6000(Sigma, M5905). Immunoprecipitations were performed using the primaryantibodies at 4° C. for 16 hours. Protein A agarose (Amersham Pharmacia)and 2 μL herrin sperm DNA (10 mg/mL) were then added to the samples for2 hours. Immunoprecipitates were then washed and heated at 65° C. for 3minutes in RIPA buffer. DNA was recovered from immunoprecipitates andinput using ethanol precipitation and phenol extraction. Atoh1 enhancerDNA was amplified using PCR and following primer pair:

(SEQ ID NO: 18) Sense: 5′-GGGGAGAGGCAGGGGAGGAGAG-3′ (SEQ ID NO: 19)Antisense: 5′-AGGCCGGGGAGGGTGACGA-3′

Samples were then analyzed using agarose gel electrophoresis. As shownin FIG. 6, Atoh1 enhancer DNA was detected in the β-catenin precipitatedsamples and input samples. Atoh1 enhancer DNA was not amplified fromcontrol chromatin immunoprecipitated with nonimmune IgG. Theseobservations suggest that β-catenin binds directly to the Atoh1enhancer.

Similar chromatin immunoprecipitation experiments were also performedusing Neuro2a cells. Such methods are as described above except thatharvested cells were pelleted for 10 minutes at 720 g at 4° C. Nucleiwere then released in a Dounce homogenizer in PBS containing proteaseinhibitors (see above) and collected at 4° C. by centrifugation at 2400g. Sheared chromatin was collected in the supernatant by centrifugation(8,000 g at 4° C. for 10 minutes) after treatment of the nuclei with theenzymatic cocktail from the ChIP-IT™ Express kit (Active Motif) for 10minutes at 37° C. 1 μg of sheared DNA was used for immunoprecipitationusing 1 μg of mouse anti-β-catenin antibody (Upstate, 05-601, 1:100),mouse anti-LEF-1 antibody (Sigma L7901) or nonimmune mouse serum(Sigma). Precipitated chromatin was recovered after reversingcross-links and the proteins were digested with proteinase K. TargetAtoh1 regulatory DNA (AF218258) was amplified by PCR using the followingprimers, which cover the entire 1.3 kB sequence in overlapping segments(as indicated)

Sense 1 (nucleotides 33-272): (SEQ ID NO: 20) ACGTTTGGCAGCTCCCTCTCAnti-sense 1: (SEQ ID NO: 21) ATAGTTGATGCCTTTGGTAGTASense 2 (nucleotides 148-434): (SEQ ID NO: 22) ATTCCCCATATGCCAGACCACAnti-sense 2: (SEQ ID NO: 23) GGCAAAGACAGAATATAAAACAAGSense 3 (nucleotides 349-609): (SEQ ID NO: 24) AATCGGGTTAGTTCTTTGAntisense 3: (SEQ ID NO: 25) ACTCCCCCTCCCTTTCTGGTASense 4 (nucleotides 501-742): (SEQ ID NO: 26) CAGGGGGAGCTGAAGGAAGAnti-sense 4: (SEQ ID NO: 27) TTTTAAGTTAGCAGAGGAGATTASense 5 (nucleotides 675-939): (SEQ ID NO: 28) CTGAGCCCCAAAGTTGTAATGTTAnti-sense 5: (SEQ ID NO: 29) TGGGGTGCAGAGAAGACTAAASense 6 (nucleotides 926-1161): (SEQ ID NO: 30) ACCCCAGGCCTAGTGTCTCCAnti-sense 6: (SEQ ID NO: 31) TGCCAGCCCCTCTATTGTCAGSense 7 (nucleotides 1094-1367): (SEQ ID NO: 32)GTGGGGGTAGTTTGCCGTAATGTG Anti-sense 7: (SEQ ID NO: 33)GGCTCTGGCTTCTGTAAACTCTGC

As shown in FIG. 2B, β-catenin and Tcf-Lef antibody immunoprecipitatedDNA at the 5′ and 3′ ends of the 1.3 kB sequence. This observationindicates that DNA in these regions has an affinity for both proteins.These sequences were not seen in control samples exposed to serum.

Atoh1 has a 1.7 kb regulatory enhancer located 3′ to its coding region.This 3′ Atoh1 enhancer is sufficient to direct expression of aheterologous reporter gene in transgenic mice (Helms et al.,Development, 127:1185-1196, 2000). To define the binding sites on themouse Atoh1 enhancer, the murine Atoh1 3′ enhancer sequence (AF218258)was searched using MatInspector (Genomatix) software. These searchesidentified two candidate binding sites for β-catenin in combination withTcf-Lef transcriptional coactivators at nucleotides 309-315 and 966-972of AF218258. To determine whether these candidate sites had bindingaffinity for β-catenin we performed DNA pulldown assays with twobiotin-labeled oligonucleotides probes, termed probe 309 and probe 966.Each of these probes contain sequence homologous to the candidate sitesat nucleotides 309-315 and 966-972 of AF218258 and surroundingnucleotides. Probe 309 spans nucleotides 297-326 and probe 966 spansnucleotides 956-985 of AF218258. The sequences of probes 309 and 966 areas follows:

Probe 309 (SEQ ID NO: 34) 5′-ATCACCCAAACA AACAAAG AGTCAGAACTT-3′Probe 966 (SEQ ID NO: 35) 5′-GTTAGGAGCCAGA AGCAAAG GGGGTGACAC-3′

Both probe 309 and 966 encode five prime termini biotin labels. Thesequences of the candidate β-catenin/Tcf-Lef binding sites (309-315 and966-972) are shown in bold.

Pulldown assays were performed as follows. Nuclei were isolated from 10⁶HEK and Neuro2a cells following mechanical disruption with a 20 gaugeneedle.

Proteins were extracted from nuclei in 200 μl RIPA buffer with freshproteinase inhibitors at 4° C. for 60 minutes. Chromatin DNA waspelleted at 14,000 g for 15 min at 4° C. and the nuclear lysate(supernatant) was collected. Biotin-labeled DNA probe (0.3 μg) with orwithout 10 μg unlabeled DNA probe was incubated with 40 μl nuclearlysate for 30 min at room temperature with gentle shake in bindingbuffer (10 mM Tris, 50 mM KCl, 1 mM DTT, 5% glycerol, pH 7.5, 40 mM 20mer poly A and poly C) with proteinase inhibitors. Probe-bound proteinswere collected with 50 μl Streptavidin magnetic beads (AmershamPharmacia). Precipitated proteins were washed five times with bindingbuffer and boiled in 50 μl 2× sample buffer (BioRad), and thesupernatant was collected for Western blotting.

Western blots were performed to detect proteins interacting with probes309 and 966. Briefly, proteins were separated on 4-12% NuPAGE Bis-Trisgels (Invitrogen) and electotransferred to 0.2 μm nitrocellulosemembranes (BioRad). The membranes were probed with mouse anti-Atoh1antibody (Developmental Studies Hybridoma Bank), anti-Lef-1/Tcf antibody(Sigma L4270), or rabbit anti-β-catenin antibody (Sigma C2206), followedby HRP-conjugated anti-mouse (Sigma), anti-goat (Santa-Cruz) oranti-rabbit (Chemicon) antibodies. The blots were processed with ECL™(Amersham Pharmacia) according to the manufacturer's instructions.

As shown in FIGS. 7A and 7B, left columns, both β-catenin and Tcf-Lef,respectively, were detected following DNA pull down using Westernblotting. As shown in the center column of FIGS. 7A and 7B, binding ofβ-catenin and Tcf-Lef to the probes was reduced by competition withunlabeled probe. As shown in the right column of FIGS. 7A and 7B,mutation of the candidate binding sites (see SEQ ID NOs: 11 and 12) ofprobes 309 and 966 also reduced binding of β-catenin and Tcf-Lef to theprobes. The sequences of the mutant 309 and 966 probes are as follows:

Mutant Probe 309 (SEQ ID NO: 36) 5′-ATCACCCAAACACATACGAAGTCAGAACTT-3′Mutant Probe 966 (SEQ ID NO: 37) 5′-GTTAGGAGCCAGAGGATCGTGGGGTGACAC-3′The sequences of the wild type probes are as follows:Wild Type Probe 309 (nucleotides 297-326) (SEQ ID NO: 38)5′-ATCACCCAAACAAACAAGAGTCAGCACTT-3′ Wild Type Probe 966 (SEQ ID NO: 39)5′-GTTAGGAGCCAGAAGCAAAGGGGGTGACTC-3′

The sequences of the mutated candidate β-catenin/Tcf-Lef binding sites(309-315 and 966-972) are shown in bold. Both mutant probes 309 and 966encode five prime termini biotin labels. Thus, probe bound proteins werecollected with Streptavidin magnetic beads (50 μL; Amersham-Pharmacia).Precipitated proteins were washed five times with binding buffer andboiled in 50 μL sample buffer. Supernatant was collected for Westernblotting with anti-β-catenin antibody and anti-Lef-1-Tcf antibody.

The precise sequences within Atoh1 that have binding affinity forβ-catenin and Tcf-Lef were identified using the above described DNApulldown. Consistent with the observation reported above in HEK cells,in Neuro2a cell, as shown in FIG. 7C, probe 309 and 966 interacted withβ-catenin and Tcf-Lef and this interaction was reduced by competitionand destroyed by mutation.

The competition and mutation assays confirm the specificity of the DNApull down assay.

These data suggest that both of the candidate binding sites identifiedin the Atoh1 enhancer region bound to β-catenin in the Tcf/Lef complex.

As shown in FIG. 7D, dominant negative Tcf4 suppressed β-catenin inducedAtoh1 expression. Furthermore, inhibition was almost complete at higherlevels, indicating that a complex between β-catenin and Tcf-Lef isrequired for activation of Atoh1 by β-catenin.

Example 5 β-Catenin Modulates the Activity of the Atoh1 Enhancer Region

To determine whether the two confirmed β-catenin binding sites on theAtoh1 enhancer increased the functional activity of the Atoh1 enhancer,we constructed multiple Atoh1 enhancer-reporter genes with intact ormutated Atoh1 3′ enhancers.

A construct with the Atoh1 3′ enhancer controlling expression of thefirefly luciferase gene (Luc) was made as follows. A BamH1/NcoI fragmentcontaining the Atoh1 3′ enhancer region (containing theβ-catenin/Tcf-Lef binding sites identified above) with a basic β-globinpromoter was excised from an Atoh1-GFP construct (Lumpkin et al., supra)and inserted into the luciferase vector, pGL3 (Promega), at BglII/NcoIin the multiple cloning region to create a Atoh1-luc vector.

A control Luc construct was made using the Atoh1-luc vector by excisingthe Atoh1 enhancer with BgII/EcoR1, followed by blunt end ligation. Allthe sequences were confirmed by sequencing.

Site-directed mutagenesis was performed using the QuickChange® IISite-Directed Mutagenesis Kit (Stratagene), according to themanufacturer's instructions. In short, the vector containing the targetgene was denatured and annealed to the oligonucleotide primers that weredesigned according to the manufacturer's instructions, with the desiredmutations in the complimentary strands. Following temperature cycling,circular DNA was generated from the template vector containing theincorporated mutagenic primers using PfuTurbo DNA polymerase, andmethylated, parental DNA was digested with Dpn1 endonuclease. Finallythe circular, nicked dsDNA was transformed into competent cells forrepair. All mutations were confirmed after amplification by sequencing.Each of the β-catenin binding sites on the Atoh1 enhancer were mutated,alone or together, in a luciferase reporter construct, as indicated inFIGS. 8A-8E.

The wild type (WT) and mutant (MUT) constructs illustrated in FIG. 8were then used to assess the functional activity of the Atoh1 enhancerby luciferase assay. Briefly, 10⁵ murine Neuro2a cells were seeded intoa 24 well plate one day before transfection. 0.125 μg Atoh1-Luciferasereporter construct, 0.125 μg Renilla-Luciferase construct with orwithout 0.25 μg β-catenin expression construct were mixed with 0.5 μplLipofectamine™ 2000 transfection reagent in 0.125 ml opti-MEM andincubated with the cells for 4 hr. Cells were lysed after 24 hr andluciferase activity was measured using the Dual Luciferase ReporterAssay System (Promega) in a TD-20/20 Luminometer (Turner Designs).

As shown in FIG. 9, β-catenin had no effect on the control luciferaseconstruct, which does not include the Atoh1 3′ enhancer. Conversely,β-catenin increased reporter gene expression from the luciferaseconstruct encoding the WT Atoh1 3′ enhancer. β-catenin mediated Atoh1 3′enhancer reporter gene expression was reduced in the presence ofβ-catenin in all mutant constructs. β-catenin mediated upregulation wasabolished in the double mutant construct.

These data indicate that β-catenin binding to the Atoh1 3′ enhancerincreases activity of the enhancer and that both of the β-cateninbinding sites are required for maximum enhancer activity.

Example 6 A Combination of Notch Signaling Inhibition and β-CateninActivity Promotes Enhanced Atoh1 Expression

Notch signaling was inhibited using a γ-secretase inhibitor (DAPT) andβ-catenin was activated using a GSK3β inhibitor (GSKi). Briefly, bonemarrow derived MSCs that exhibit increased Atoh1 activity following theinhibition of Notch signaling were exposed to a γ-secretase inhibitorand a GSK3β inhibitor. Altered Notch activity was confirmed using CBF-1luciferase reporter.

Mesenchymal stem cells (MSCs) were isolated from human bone marrow usingthe methods described by Jeon et al. (Mol. Cell. Neurosci., 34(1):59-68,(2007)). Cells were expanded once before use and cultured in MEM-a cellculture media (Sigma-Aldrich) supplemented with 9% horse serum, 9% fetalcalf serum, and penicillin (100 U/mL) and streptomycin (100 μg/mL).

As shown in FIG. 10A, β-catenin expression was increased in cellsexposed to γ-secretase inhibitor. Furthermore, as noted above, Atoh1expression is increased by β-catenin. As shown in FIG. 10A, Atoh1expression is further increased by a combination of β-catenin and Notchinhibition. To confirm the role of β-catenin in this observation,β-catenin expression was modulated using siRNA (as described in Example3 above). The decrease in β-catenin is shown in FIG. 10B. As shown inFIG. 10C, suppression of β-catenin prevented any β-catenin expressionfollowing γ-secretase treatment and reduced Atoh1 expression. Similarresults were also observed when Notch signaling was inhibited usingnon-γ-secretase inhibitors (see FIG. 10D). This result demonstrates therelationship between the inhibition of Notch signaling and β-cateninactivity is not limited to the use of γ-secretase inhibitors.

Disruption of β-catenin mediated transcription by overexpression ofdominant negative Tcf (dn Tcf) also reversed the increase in Atoh1expression observed in cells treated with the inhibitor of Notchsignaling (see FIG. 10E). Conversely, whereas β-catenin and Atoh1expression were diminished following the elevation of Notch signaling,activation of β-catenin by Wnt3a rescued Atoh1 expression (see FIG.10E).

These results suggest that the inhibition of Notch signaling combinedwith β-catenin activity may function synergistically to increase Atoh1expression. Accordingly, combined therapy using Notch signalinginhibition and a β-catenin modulating compound, such as a β-cateninagonist, can be used to promote Atoh1 expression.

Example 7 β-Catenin Promotes the Conversion of Inner Ear Stem Cells toAtoh1 Positive Cells in Transgenic Mice

Mice that express nuclear green fluorescent protein (GFP) under thecontrol of the Atoh1 enhancer (Atoh1-nGFP mice (Lumpkin et al., supra))were used to assess the conversion of inner ear stem cells into haircells. Increased expression of GFP in these animals indicates anincrease in the activity of the Atoh1 3′ enhancer. Inner ear stem cellsderived from the transgenic animals were transduced with adenovirusescontaining β-catenin or GFP under the control of a CMV promoter(Michiels et al., Nat. Biotec., 20:1154-1157, 2002).

Inner ear stem cells were isolated from Atoh1-nGFP as previouslydescribed (Li et al., Nat. Med., 9:1293-1299, 2003). Briefly utricleswere dissected from 4 Atoh1-nGFP mice at postnatal day four (P4) andwere trypsinized into a single cell suspension. The released cells werethen grown in suspension for seven days in DMEM/FD12 medium (1:1)supplemented with N2/B27, 10 ng/mL FGF-2 (Chemicon), 50 ng/mL IGF(Chemicon), and 20 ng/mL EGF (Chemicon) to obtain spheres.

Inner ear stem cells isolated as spheres were seeded into afour-compartment 35 mm tissue culture dish and grown as a monolayer inDMEM/N2 medium. 10⁶ cells were infected with β-catenin, GFP or emptyadenoviruses (9×10⁷ viral particles) in 100 μL Opti-MEM for 16 hours.

As shown in FIG. 11A, transduction of inner ear stem cells with thecontrol GFP adenovirus resulted in GFP expression in 68% of the cells.As shown in FIG. 5C, transduction of inner ear stem cells with β-cateninadenovirus increased the number of Atoh1 positive cells compared toinner ear stem cells transduced with empty virus. This data suggeststhat β-catenin increased Atoh 1 activity in inner ear stem cellsobtained from Atoh1-nGFP mice. This observation is consistent with thedifferentiation of inner ear stem cells to hair cells. To furtherconfirm the differentiation of the inner ear stem cells to hair cells,transduced cells were analyzed using immunocytochemistry to detect haircell specific markers. Immunostaining was performed using rabbitantibody to myosin VII1 (Proteus Bioscience) at a 1:1000 dilution ormouse monoclonal antibody PC10 to detect PCNA (eBioscience) at a 1:100dilution. Positively stained cells were counted using MetaMorph Imaging7.0 and statistics were performed from three independent experiments.

As shown in FIG. 11D, when 5000 cells were counted in three independentexperiments, the number of cells staining positive for Atoh1 and myosinVIIa doubled in cells expressing β-catenin (Atoh1 positive cellsincreased from 8.9% to 15.8% and myosin VIIa positive cells increasedfrom 3.3% to 6.6%). As Atoh1 and myosin VIIa are known specific haircell markers, this observation confirms that β-catenin promotes thedifferentiation of inner ear stem cells into hair cells. To correlateβ-catenin overexpression with the conversion of inner ear progenitorcells into Atoh1 positive cells, an expression vector encoding theβ-catenin coding region followed by the reporter sequence IRES-DsRed wasconstructed.

The β-catenin-IRES-DsRed construct was by cloning human β-catenin cDNAcontaining Xba I (enzymes from New England Biolabs) sticky ends intopIRES2-DsRed Express (Clontech) at the Nhe I site.

Inner ear stem cells isolated and seeded as described above wheretransfected with 4 μg IRES-DsRed empty vector or 4 μgβ-catenin-IRES-DsRed using 3 μL Lipofectamine™ 2000 transfection reagentin 100 μL Opti-MEM for 4 hours. Transfected cells were then analyzed byimmunocytochemistry after 5 days. Immunostaining was performed asdescribed above.

As shown in FIG. 12B and Table 1, none of the 14 cells expressingIRES-DsRed empty vector stained positively for Atoh1. In contrast, asshown in FIG. 12A and table 1, 8 of 15 cells expressingβ-catenin-IRES-DsRed stained positively for Atoh1.

TABLE 1 Quantification of β-Catenin-Mediated Atoh1 Expression in InnerEar Stem Cells DsRed Atoh1 Positive Positive Cells Cells Transfection(Red) (Green) β-catenin-IRES-DsRed 14 8 IRES-DsRed 15 0 N = 3; cellscounted = 1000.

To ascertain whether the increase in Atoh1 positive cells observed abovewas due to increased proliferation of the inner ear stem cells, as hasbeen reported for other neural progenitor cells (Adachi et al., StemCells, 25:2827-36, 2007; Woodhead et al., J. Neurosci., 26:12620-12630,2006), labeling for PCNA was assessed in β-catenin expressing cells.

Adenovirus mediated β-catenin expression resulted in 68±7.9% PCNApositive cells, which was not significantly different (p>0.05) fromcells transduced with empty adenovirus (69.7±5.2% PCNA positive cells)and non-transduced cells (72±8.8% PCNA positive cells) based on threeindependent experiments in which 5000 cells were counted.

This data suggests that cell proliferation was not required forβ-catenin mediated cell differentiation.

Example 8 β-Catenin Mediated Hair Cell Formation

As shown in FIG. 13, β-catenin expression promoted the formation ofextra rows of outer hair cells at E16. 8.1 E+07 adenovirus particleswere applied to organ of Corti dissected from E16 Atoh1-nGFP embryos andcultured for 5 days. Adenovirus encoding As shown in FIGS. 13C and 12D,β-catenin increased the number of Atoh1 positive outer hair cellscompared to untreated (A) or cells treated with empty adenovirus (B).

As shown in FIG. 14 and table 2, 8.1E+07 adenovirus particles were usedto infect organs of Corti dissected from E16 Atoh1-nGFP embryos thatwere then cultured for 5 days. Images were captured prior to infected(see FIG. 13B) and 5 days post-infected (see FIG. 13A). The results werequantified and are shown in Table 2. As shown in Table 2, a 32±3.1%increase was observed following treatment.

TABLE 2 Treatment with Ad-β-catenin Pre-infected Post-infection IHC 256249 OHC 768 1014 N = 3.

Example 9 Assessment of the Combined Affect of β-Catenin and Inhibitorsof the Notch Signaling Pathway on the Conversion of Inner Ear Stem Cellsto Atoh1 Positive Cells in Transgenic Mice

Mice that express nuclear green fluorescent protein (GFP) under thecontrol of the Atoh1 enhancer (Atoh1-nGFP mice (Lumpkin et al., supra))were processed as described in Example 9.

Inner ear stem cells isolated as spheres were seeded into afour-compartment 35 mm tissue culture dish and grown as a monolayer inDMEM/N2 medium. 10⁶ cells were infected with combinations of a β-cateninadenovirus or one or more β-catenin modulating compounds, GFPadenovirus, empty adenoviruses (9×10⁷ viral particles) and an inhibitorof the Notch signaling pathway in 100 μL Opti-MEM for 16 hours, as shownin Table 3 (X indicates cells are treated):

TABLE 3 Well GFP Empty Notch No. β-catenin adenovirus Adenovirusinhibitor 1 X 2 X 3 X 4 X 5 X X 6 X X 7 X X

Following treatment, cells were analyzed for Atoh1 and myosin VIIaexpression.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1.-43. (canceled)
 44. A method of treating a subject who has hearingloss as a result of loss of auditory hair cells, the method comprising:identifying a subject who has hearing loss as a result of loss ofauditory hair cells; and administering to the middle or inner ear of thesubject a composition comprising a glycogen synthase kinase 3β (GSK3β)inhibitor and a proteasome inhibitor in an amount effective to increasethe number of auditory hair cells in the subject; thereby treating thehearing loss as a result of loss of auditory hair cells in the subject.45. The method of claim 44, wherein the subject has sensorineuralhearing loss, auditory neuropathy, or both, as a result of loss ofauditory hair cells.
 46. The method of claim 44, wherein the compositionfurther comprises a β-catenin polypeptide.
 47. The method of claim 44,wherein the composition is injected into the luminae of the cochlea. 48.The method of claim 44, wherein the one or more GSK3β inhibitors isselected from the group consisting of lithium chloride, purvalanol A,olomoucine, alsterpaullone, kenpaullone,benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione,2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4 ]-oxadiazole,2,4-dibenzyl-5-oxothiadiazolidine-3-thione,(2′Z,3′E)-6-bromoindirubin-3′-oxime (BIO), α 4 dibromoacetophenone,2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone,N-(4-nethoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea,4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione,2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole,2,4-dibenzyl-5-oxothiadiazolidine-3-thione, α-4-dibromoacetophenone,2-chloro-1-(4, 5-dibromo-thiophen-2-yl)-ethanone, andN-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea.
 49. The method ofclaim 44, wherein the one or more GSK3β inhibitor is an indirubin. 50.The method of claim 49, wherein the indirubin is selected from the groupconsisting of: indirubin-5-sulfonamide, indirubin-5-sulfonic acid(2-hydroxyethyl)-amide, indirubin-3′-monoxime;5-iodo-indirubin-3′-monoxime, 5-fluoroindirubin, 5, 5′-dibromoindirubin,5-nitroindirubin, 5-chloroindirubin, 5-methylindirubin, and5-bromoindirubin.
 51. The method of claim 44, wherein the methodcomprises administering the composition to the middle ear of thesubject.
 52. The method of claim 44, wherein the method comprisesadministering the composition to the inner ear of the subject.
 53. Themethod of claim 48, wherein the GSK3β inhibitor is(2′Z,3′E)-6-bromoindirubin-3′-oxime (BIO).
 54. The method of claim 44,wherein the proteasome inhibitor is selected from the group consistingof Bortezomib, MG132, lactacystin, and proteasome inhibitor PSI.
 55. Themethod of claim 44, wherein the auditory hair cell expresses atonalprotein homologue 1 (Atoh1).
 56. The method of claim 44, wherein thecomposition further comprises an inhibitor of the Notch signalingpathway.
 57. The method of claim 56, wherein the inhibitor of the Notchsignaling pathway is a γ-secretase inhibitor.